Dna sequencing by synthesis using modified nucleotides and nanopore detection

ABSTRACT

This disclosure is related to a method of sequencing a single-stranded DNA using deoxynucleotide polyphosphate analogues and translocation of tags from incorporated deoxynucleotide polyphosphate analogues through a nanopore.

This application claims priority of U.S. Provisional Application No.61/424,480, filed Dec. 17, 2010, and U.S. Provisional Application No.61/557,558, filed Nov. 9, 2011, the content of each of which is herebyincorporated by reference in its entirety.

Throughout this application, certain patents and publications arereferenced, the latter by authors and publication year. Full citationsfor these publications may be found immediately preceding the claims.The disclosures of these patents and publications in their entiretiesare hereby incorporated by reference into this application in order todescribe more fully the state of the art to which this inventionrelates.

BACKGROUND OF THE INVENTION

DNA sequencing is a fundamental technology for biology. Severalanalytical methods have been developed to detect DNA or RNA at singlemolecule level using chemical or physical microscopic technologies[Perkins et al. 1994, Rief et al. 1999, Smith et al. 1996, andVercoutere et al. 2001].

In the past few years, ion-sensing technologies such as ion channel,which relies on the detection of hydrogen ion (H⁺) released when anucleotide is incorporated into a strand of DNA by a polymerase[Rothberg et al. 2011], have been explored to detect individual DNA orRNA strands [Kasianowicz 2003 & 2004, Chandler et al. 2004, Deamer etal. 2002, Berzukov et al. 2001, and Henrickson et al. 2000].

It has been demonstrated that an α-hemolysin channel, an exotoxinsecreted by a bacterium, can be used to detect nucleic acids at thesingle molecule level [Kasianowicz et al. 1996]. An α-hemolysin proteinis a monomeric polypeptide which self-assembles in a lipid bilayermembrane to form a heptameric pore, with a 2.6 nm-diameter vestibule and1.5 nm-diameter limiting aperture (the narrowest point of the pore)[Meller et al. 2000, Akeson et al. 1999, and Deamer et al. 2002]. Thelimiting aperture of the nanopore allows linear single-stranded but notdouble-stranded, nucleic acid molecules (diameter ˜2.0 nm) to passthrough. In an aqueous ionic salt solution such as KCl, when anappropriate voltage is applied across the membrane, the pore formed byan α-hemolysin channel conducts a sufficiently strong and steady ioniccurrent. The polyanionic nucleic acids are driven through the pore bythe applied electric field, thus blocking or reducing the ionic currentthat would be otherwise unimpeded. This process of passage generates anelectronic signature (FIG. 1) [Vercoutere et al. 2001 and Deamer et al.2002]. A particular nucleic acid molecule, when entering and passingthrough the nanopore generates a characteristic signature thatdistinguishes it from other nucleic acid molecules. The duration of theblockade is proportional to the length of nucleic acid, and the signalstrength is related to the steric and electronic properties of thenucleotides, namely the identity of the four bases (A, C, G and T). Thusa specific event diagram, which is a plot of translocation time versusblockade current, is obtained and used to distinguish the length and thecomposition of polynucleotides by single-channel recording techniquesbased on characteristic parameters such as translocation current,translocation duration, and their corresponding dispersion in thediagram [Meller et al. 2000].

It has also been shown that a protein nanopore with a covalentlyattached adaptor can accurately identify unlabeled nucleoside5′-monophosphates (dAMP, dGMP, dCMP & dTMP) with high accuracy [Clarkeet al. 2009]. For example, aminocyclodextrin adaptor has been covalentlyattached within the α-hemolysin pore successfully. When a dNMP iscaptured and driven through the pore in a lipid bilayer membrane, theionic current through the pore is reduced to one of four levels, eachrepresenting one of the four dNMP's (A, G, C, or T). Moreover, Robertsonet al. [2007] have recently demonstrated that when a poly(ethyleneglycol) (PEG) molecule enters a single α-hemolysin pore, it causesdistinct mass-dependent conductance states with characteristic meanresidence times. The conductance-based mass spectrum clearly resolvesthe repeat units of ethylene glycol, and the residence time increaseswith the mass of the PEG.

Although the current nanopore approach shows promise as a DNA detectionmethod, the more demanding goal of accurate base-to-base sequencing hasnot yet been achieved.

SUMMARY OF THE INVENTION

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, with a DNA        polymerase and four deoxyribonucleotide polyphosphate (dNPP)        analogues at least one of which can hybridize with each of an A,        T, G, or C nucleotide in the DNA being sequenced under        conditions permitting the DNA polymerase to catalyze        incorporation of one of the dNPP analogues into the primer if it        is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each of the four dNPP analogues has the        structure:

-   -    wherein the base is adenine, guanine, cytosine, thymine or        uracil, or a derivative of one or more of these bases, wherein        R₁ is OH, wherein R₂ is H, wherein X is O, NH, S or CH₂, wherein        n is 1, 2, 3, or 4, wherein Z is O, S, or BH₃, and with the        proviso that (i) the type of base on each dNPP analogue is        different from the type of base on each of the other three dNPP        analogues, and (ii) either the value of n of each dNPP analogue        is different from the value of n of each of the other three dNPP        analogues, or the value of n of each of the four dNPP analogues        is the same and the type of tag on each dNPP analogue is        different from the type of tag on each of the other three dNPP        analogues, wherein incorporation of the dNPP analogue results in        release of a polyphosphate having the tag attached thereto; and    -   (b) identifying which dNPP analogue has been incorporated into        the primer to form a DNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, whichever is applicable, thereby        permitting identifying the nucleotide residue in the        single-stranded DNA complementary to the incorporated dNPP        analogue; and    -   (c) repeatedly performing step (b) for each nucleotide residue        of the single-stranded DNA being sequenced, wherein in each        repetition of step (b) the dNPP analogue is incorporated into        the DNA extension product if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,        thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, a DNA        polymerase and a deoxyribonucleotide polyphosphate (dNPP)        analogue under conditions permitting the DNA polymerase to        catalyze incorporation of the dNPP analogue into the primer if        it is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein the dNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, or a derivative of each thereof, wherein R₁ is —OH,        —O—CH₂N₃ or —O-2-nitrobenzyl, wherein R₂ is H, wherein X is O,        NH, S or CH₂, wherein n is 1, 2, 3, or 4, wherein Z is O, S, or        BH₃, and    -    wherein if the dNPP analogue is not incorporated, iteratively        repeating the contacting with a different dNPP analogue until a        dNPP analogue is incorporated, with the proviso that (1) the        type of base on each dNPP analogue is different from the type of        base on each of the other dNPP analogues, and (2) either the        value of n of each dNPP analogue is different from the value of        n of each of the other three dNPP analogues, or the value of n        of each of the four dNPP analogues is the same and the type of        tag on each dNPP analogue is different from the type of tag on        each of the other three dNPP analogues,    -    wherein incorporation of a dNPP analogue results in release of        a polyphosphate having the tag attached thereto;    -   (b) determining which dNPP analogue has been incorporated into        the primer to form a DNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded DNA complementary to        the incorporated dNPP analogue;    -   (c) repeatedly performing steps (a) and (b) for each nucleotide        residue of the single-stranded DNA being sequenced, wherein in        each repetition of step (a) the dNPP analogue is incorporated        into the DNA extension product if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,        thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, with a DNA        polymerase and at least four deoxyribonucleotide polyphosphate        (dNPP) analogues under conditions permitting the DNA polymerase        to catalyze incorporation of one of the dNPP analogues into the        primer if it is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each of the four dNPP analogues has a structure        chosen from the following:

-   -    wherein the base is adenine, guanine, cytosine, thymine or        uracil, or a derivative of each thereof, wherein Y is a tag,        wherein R₁, if present, is OH, wherein R₂, if present, is H,        wherein X is a cleavable linker, wherein Z is O, S or BH₃,        wherein n is 1, 2, 3, or 4, wherein A is O, S, CH₂, CHF, CFF, or        NH, and with the proviso that (i) the type of base on each dNPP        analogue is different from the type of base on each of the other        three dNPP analogues, and (ii) the type of tag on each dNPP        analogue is different from the type of tag on each of the other        three dNPP analogues;    -   (b) cleaving the tag from the dNPP analogue incorporated in step        (a); and    -   (c) determining which dNPP analogue was incorporated in step (a)        by applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from tag cleaved        off in step (b) translocating through the nanopore, wherein the        electronic change is different for each different type of tag,        thereby identifying the nucleotide residue in the        single-stranded DNA complementary to the incorporated dNPP        analogue; and    -   (d) repeatedly performing steps (a), (b) and (c) for each        nucleotide residue of the single-stranded DNA being sequenced,        wherein in each repetition of step (a) the dNPP analogue is        incorporated into the DNA extension product resulting from the        previous iteration of step (a) if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,        thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane, wherein the single-stranded DNA        has a primer hybridized to a portion thereof, a DNA polymerase        and a deoxyribonucleotide polyphosphate (dNPP) analogue under        conditions permitting the DNA polymerase to catalyze        incorporation of the dNPP analogue into the primer if it is        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to a nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product,        wherein the dNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, or a derivative of each thereof, wherein Y is a tag,        and wherein R₁ if present is OH, —OCH₂N₃ or —O-2-nitrobenzyl, R₂        if present is H, wherein X is a cleavable linker, wherein Z is        O, S or BH₃, wherein n is 1, 2, 3, or 4, wherein A is 0, S, CH₂,        CHF, CFF, or NH,    -    and if the dNPP analogue is not incorporated, iteratively        repeating the contacting with a different dNPP analogue until a        dNPP analogue is incorporated, with the proviso that (1) the        type of base on each dNPP analogue is different from the type of        base on each other dNPP analogue, and (2) the type of tag on        each dNPP analogue is different from the type of tag on each        other dNPP analogue,    -    wherein incorporation of a dNPP analogue results in release of        a polyphosphate having the tag attached thereto;    -   (b) cleaving the tag from the dNPP analogue incorporated in step        (a); and    -   (c) determining which dNPP analogue was incorporated in step (a)        to form a DNA extension product by applying a voltage across the        membrane and measuring an electronic change across the nanopore        resulting from the tag cleaved off in step (b) translocating        through the nanopore, wherein the electronic change is different        for each type of tag, thereby identifying the nucleotide residue        in the single-stranded DNA complementary to the incorporated        dNPP analogue;    -   (d) iteratively performing steps (a) through (c) for each        nucleotide residue of the single-stranded DNA being sequenced,        wherein in each iteration of step (a) the dNPP analogue is        incorporated into the DNA extension product resulting from the        previous iteration of step (a) if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,        thereby determining the nucleotide sequence of the        single-stranded DNA.

A process for producing a nucleotide triphosphate analogue, wherein thenucleotide triphosphate analogue differs from a nucleotide triphosphateby having a tag attached to the terminal phosphate thereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        dicyclohexylcarbodiimide/dimethylformamide under conditions        permitting production of a cyclic trimetaphosphate;    -   b) contacting the product resulting from step a) with a tag        having a hydroxyl or amino group attached thereto under        conditions permitting nucleophilic opening of the cyclic        trimetaphosphate so as to bond the tag to a terminal phosphate        thereby forming the nucleotide triphosphate analogue.

A process for producing a nucleotide triphosphate analogue, wherein thenucleotide triphosphate analogue differs from a nucleotide triphosphateby having a tag attached to the terminal phosphate thereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        dicyclohexylcarbodiimide/dimethylformamide under conditions        permitting production of a cyclic trimetaphosphate;    -   b) contacting the product resulting from step a) with a        nucleophile so as to form an —OH or —NH₂ functionalized        compound;    -   c) reacting the product of step b) with a tag having a —COR        group attached thereto under conditions permitting the tag to        bond indirectly to a terminal phosphate thereby forming the        nucleotide triphosphate analogue.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a monophosphate group attached thereto under conditions        permitting formation of the nucleotide tetraphosphate analogue.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with phosphoric        acid under conditions permitting formation of a nucleotide        tetraphosphate;    -   c) contacting the nucleotide tetraphosphate with 1)        carbonyldiimidazole/dimethylformamide; 2) a nucleophile and        then 3) NH₄OH so as to form an —OH or —NH₂ functionalized        compound;    -   d) contacting the product of step c) with a tag having a —COR        group attached thereto under conditions permitting the tag to        bond indirectly to a terminal phosphate thereby forming the        nucleotide tetraphosphate analogue.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -   b) contacting the product resulting from step a) with phosphoric        acid under conditions permitting formation of a nucleotide        tetraphosphate;    -   c) contacting the nucleotide tetraphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form a        compound having the structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine.

A process for producing a nucleotide pentaphosphate analogue, whereinthe nucleotide pentaphosphate analogue differs from a nucleotidepentaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a pyrophosphate group attached thereto under conditions        permitting formation of the nucleotide pentaphosphate analogue.

A process for producing a nucleotide pentaphosphate analogue, whereinthe nucleotide pentaphosphate analogue differs from a nucleotidepentaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a        pyrophosphate group under conditions permitting formation of a        nucleotide pentaphosphate;    -   c) contacting the nucleotide pentaphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form the        nucleotide pentaphosphate analogue.

A process for producing a nucleotide hexaphosphate analogue, wherein thenucleotide hexaphosphate analogue differs from a nucleotidehexaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a triphosphate group attached thereto under conditions        permitting formation of the nucleotide hexaphosphate analogue.

A process for producing a nucleotide hexaphosphate analogue, wherein thenucleotide hexaphosphate analogue differs from a nucleotidehexaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a        triphosphate group under conditions permitting formation of a        nucleotide hexaphosphate;    -   c) contacting the nucleotide hexaphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form the        nucleotide hexaphosphate analogue.

A compound having the structure:

wherein the tag is ethylene glycol, an amino acid, a carbohydrate, adye, mononucleotide, dinucleotide, trinucleotide, tetranucleotide,pentanucleotide or hexanucleotide, wherein R₁ is OH, wherein R₂ is H orOH, wherein X is O, NH, S or CH₂, wherein Z is O, S, or BH₃, wherein thebase is adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine ora 5-methylpyrimidine, and wherein n is 1, 2, 3, or 4.

A compound having the structure:

wherein in each structure n is, independently, 1, 2, 3 or 4, and m is,independently, an integer from 0 to 100, and wherein when m is 0 theterminal phosphate of the dNPP is bonded directly to the 3′ O atom ofthe nucleoside shown on the left hand side of the structure, wherein R₁is —OH, or —O—CH₂N₃, and R₂ is H or OH.

A composition comprising at least four deoxynucleotide polyphosphate(dNPP) analogues, each having a structure selected from the structuresset forth in claims 74 and 75, wherein each of the four dNPP analoguescomprises a type of base different from the type of base of the otherthree dNPP analogues.

A compound having the structure:

wherein m an integer from 0 to 100, and wherein the compound comprises asingle type of base, and wherein the base is adenine, guanine, cytosine,uracil or thymine or a derivative thereof of each.

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine.

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine, and R is a substituted orunsubstituted hydrocarbyl, up to 3000 daltons.

A compound having the structure:

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine, and m is an integer from 1-50.

A compound having the structure:

wherein n is 1 or 2 and the base is adenine, guanine, cytosine, thymine,uracil, a 7-deazapurine or a 5-methylpyrimidine.

A compound having the structure:

wherein R₁ is —OH, or —O—CH₂N₃, and R₂ is H or OH.

A method for determining the nucleotide sequence of a single-strandedRNA comprising:

-   -   (a) contacting the single-stranded RNA, wherein the        single-stranded RNA is in an electrolyte solution in contact        with a nanopore in a membrane, wherein the single-stranded RNA        has a primer hybridized to a portion thereof, with a RNA        polymerase and at least four ribonucleotide polyphosphate (rNPP)        analogues under conditions permitting the RNA polymerase to        catalyze incorporation of one of the rNPP analogues into the        primer if it is complementary to the nucleotide residue of the        single-stranded RNA which is immediately 5′ to a nucleotide        residue of the single-stranded RNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a RNA extension        product, wherein each of the four rNPP analogues has the        structure:

-   -    wherein the base is adenine, guanine, cytosine, thymine or        uracil, or a derivative thereof of each, wherein R₁ is OH,        wherein R₂ is OH, wherein X is O, NH, S or CH₂, wherein n is 1,        2, 3, or 4, wherein Z is O, S, or BH₃, and with the proviso        that (i) the type of base on each rNPP analogue is different        from the type of base on each of the other three rNPP analogues,        and (ii) either the value of n of each rNPP analogue is        different from the value of n of each of the other three rNPP        analogues, or the value of n of each of the four rNPP analogues        is the same and the type of tag on each rNPP analogue is        different from the type of tag on each of the other three rNPP        analogues, wherein incorporation of the rNPP analogue results in        release of a polyphosphate having the tag attached thereto; and    -   (b) determining which rNPP analogue has been incorporated into        the primer to form a RNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded RNA complementary to        the incorporated rNPP analogue; and    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded RNA being sequenced, wherein in        each iteration of step (a) the rNPP analogue is incorporated        into the RNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded RNA which is immediately 5′ to a        nucleotide residue of the single-stranded RNA hybridized to the        3′ terminal nucleotide residue of the RNA extension product,        thereby determining the nucleotide sequence of the        single-stranded RNA.

A method for determining the nucleotide sequence of a single-strandedRNA comprising:

-   -   (a) contacting the single-stranded RNA, wherein the        single-stranded RNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        RNA has a primer hybridized to a portion thereof, a RNA        polymerase and a ribonucleotide polyphosphate (rNPP) analogue        under conditions permitting the RNA polymerase to catalyze        incorporation of the rNPP analogue into the primer if it is        complementary to the nucleotide residue of the single-stranded        RNA which is immediately 5′ to a nucleotide residue of the        single-stranded RNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a RNA extension product,        wherein the rNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, wherein R₁ is —OH, —O—CH₂N₃ or —O-2-nitrobenzyl,        wherein R₂ is —OH, wherein X is O, NH, S or CH₂, wherein n is 1,        2, 3, or 4, wherein Z is O, S, or BH₃,    -    and wherein if the rNPP analogue is not incorporated,        iteratively repeating the contacting with a different rNPP        analogue until a rNPP analogue is incorporated, with the proviso        that (1) the type of base on each rNPP analogue is different        from the type of base on each of the other rNPP analogues,        and (2) either the value of n of each rNPP analogue is different        from the value of n of each of the other three rNPP analogues,        or the value of n of each of the four rNPP analogues is the same        and the type of tag on each rNPP analogue is different from the        type of tag on each of the other three rNPP analogues,    -    wherein incorporation of a rNPP analogue results in release of        a polyphosphate having the tag attached thereto;    -   (b) determining which rNPP analogue has been incorporated into        the primer to form a RNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or different        for each type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded RNA complementary to        the incorporated dNPP analogue;    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded RNA being sequenced, wherein in        each iteration of step (a) the rNPP analogue is incorporated        into the RNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded RNA which is immediately 5′ to a        nucleotide residue of the single-stranded RNA hybridized to the        3′ terminal nucleotide residue of the RNA extension product,        thereby determining the nucleotide sequence of the        single-stranded RNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. α-Hemolysin protein self-assembles in a lipid bilayer to form anion channel and a nucleic acid stretch passes through it (top), with thecorresponding electronic signatures generated (bottom) [Vercoutere etal. 2001 and Deamer et al. 2002].

FIG. 2. Structures of nucleotides deoxyribonucleotide adenosinetriphosphate, deoxyribonucleotide guanosine triphosphate,deoxyribonucleotide cytosine triphosphate, and deoxyribonucleotidethymidine triphosphate.

FIG. 3. Mechanism of primer extension and release oftagged-polyphosphate for detection.

FIG. 4. Structure of four phosphate-tagged nucleoside-5′-polyphosphates.

FIG. 5. Synthesis of phosphate-tagged nucleoside-5′-triphosphates.

FIG. 6. Synthesis of phosphate-tagged nucleoside-5′-tetraphosphates.

FIG. 7. Synthesis of terminal phosphate-taggednucleoside-5′-pentaphosphates.

FIG. 8. a) oligo-3′ to 5′-phosphate attachment, b) oligo-5′ to5′-phosphate attachment, c) detectable moiety after polymerase reaction.

FIG. 9(A). Synthesis of base-modified nucleoside-5′-triphosphates. FIG.9(B). Cleavage of base-modified nucleoside-5′-triphosphate and cleavagewith TCEP.

FIG. 10. Synthesis of 3′-O-modified nucleoside-5′-triphosphates. A.3′-O-2-nitrobenzyl attached dNTPs; B. 3′-O-azidomethyl attached dNTPs;C. Detectable moiety after polymerase extension and TCEP cleavage; andD. Detectable moiety after polymerase extension and UV cleavage.

FIG. 11. DNA extension reaction using phosphate modified nucleotideanalogues.

FIG. 12. DNA extension reaction using base-tagged nucleotide analogues.

FIG. 13. DNA extension reaction using 2′- or 3′-OH labeled nucleotideanalogues.

FIG. 14. Schematic of DNA sequencing by nanopore with modifiednucleotides, particularly applicable to single molecule real timesequencing involving addition of all 4 nucleotides and polymerase atsame time to contact a single template molecule.

FIG. 15. Phosphate, Base, 2′- and 3′-modified nucleoside phosphates withpossible linkers and tags

-   -   BASE=adenine, guanine, thymine, cytosine, uracil, 5-methyl C,        7-deaza-A, 7-deaza-G or their derivatives thereof;    -   R₁ and R₂=H, OH, F, NH₂, N₃, or OR′;    -   n=1-5;    -   A=O, S, CH₂, CHF, CFF, NH;    -   Z=O, S, BH₃;    -   X=Linker which links phosphate or the 2′-0 or 3′-0 or the base        to the detectable moiety and may contain 0,

N or S, P atoms. (The linker can also be a detectable moiety, directlyor indirectly, such as amino acids, peptides, proteins, carbohydrates,PEGs of different length and molecular weights, organic or inorganicdyes, fluorescent and fluorogenic dyes, drugs, oligonucleotides, masstags, chemiluminescent tags and may contain positive or negativecharges);

-   -   Y=tags or detectable moiety, such as aliphatic or organic        aromatic compounds with one or more rings, dyes, proteins,        carbohydrates, PEGs of different length and molecular weights,        drugs, oligonucleotides, mass tags, fluorescent tags,        chemiluminescent tags and may contain positive or negative        charge.

FIG. 16. Structures of PEG-phosphate-labeled nucleotides and examples ofpossible PEGs with different reactive groups to react with functionalgroups.

FIG. 17. Non-limiting, specific examples of reactive groups on theterminal phosphates, which can also be attached with appropriate changesto a nucleoside base moiety, and groups with which groups can react toform tags.

FIG. 18. A schematic of array of nanopores for massive parallel DNAsequencing by synthesis.

FIG. 19. Synthesis of PEG-phosphate-labeled nucleotides.

FIG. 20. MALDI-TOF mass spectra of the DNA extension products generatedby incorporation of PEG-phosphate-labeled nucleotide analogues(dG4P-PEG). The single products shown in the spectra indicate that thedG4P-PEG24 and dG4P-PEG37 are incorporated at nearly 100% efficiency.

FIG. 21. The relative blockade depth distributions for α-hemolysinnanopore in the presence of PEGs that contain either 49, 37, 24, or 16ethylene oxide monomers at +40 mV applied potential. The four speciesare easily identified.

FIG. 22. (A) Separation and mass distribution of mixed poly (ethyleneglycol) (PEG) units through a single nanopore; and (B) selection of 4distinct PEG units with base line separation as tags for the 4 bases, A,C, G, and T. The structures of linear and branched PEGs are also shown.

FIG. 23. Synthesis of charged PEG-triphosphates (the charge can beadjusted based on the requirements).

FIG. 24. Synthesis of phosphate-tagged nucleoside-5′-triphosphates.

FIG. 25. Synthesis of phosphate-tagged nucleoside-5′-tetraphosphates

FIG. 26. Synthesis of terminal phosphate-taggednucleoside-5′-pentaphosphates.

FIG. 27. CMOS-integrated nanopore measurement platform: (A) a micrographof the eight-channel CMOS preamplifier chip with an image of oneamplifier channel with the integrated cis-side electrode; (B) diagramshowing the two-chip integration with a solid-state nanopore; (C)diagram showing the cross section of the chip and how the nanopore isetched directly into the chip in the one-chip implementation; packagingoccurs with an independent well on the cis side; and a TEM image of a3.5-nm-diameter nanopore.

FIG. 28. Electrical performance of the CMOS-integrated nanoporeelectronics (A) Input-referred baseline current noise spectrum forC_(F)=0.15 pF, 1 MHz 4-pole Bessel filter, f_(s)=4MS/s. Also shown isthe measured open-headstage of an Axopatch 200B in whole-cell mode withμβ=1, 100 kHz 4-pole Bessel filter, f_(s)=250 kS/s. (B) Noise floor ofthe new amplifier with a nanopore attached compared with the samenanopore measured by the Axopatch 200B.

FIG. 29. Tethering of the polymerase in the vicinity of the nanopore. Awell helps to restrict diffusion. L denotes the critical distance fromthe pore opening at which molecular motions due to diffusion andelectrophoresis are equal.

FIG. 30. Synthesis of Tag-labeled-nucleoside-5′-polyphosphates.

FIG. 31. Synthesis of 3′-O-blocked-PEG-nucleotides.

FIG. 32. Sequencing by synthesis with PEG-nucleotides and nanoporedetection (many copies of the same DNA molecule immobilized on a beadand addition of one PEG-nucleotide at a time). Use same PEGattached tothe all four nucleotides. Add one PEG-nucleotide at a time, reads atleast one base per cycle if correct nucleotide is incorporated.

FIG. 33. Sequencing by synthesis with 3′-O-blocked-PEG-nucleotides andnanopore detection (many copies of same DNA molecule immobilized on abead and addition of all four 3′-O-blocked-PEG-nucleotides). Add allfour 3′-blocked, different size PEG attached nucleotides (3′=blockeddNTP-PEGs) together. Detection of the incorporated nucleotide based onthe blockade signal of the released PEGs. The 3′-blocking group isremoved by TECP treatment and continue cycle for correctly sequence thetemplate including homopolymeric regions.

FIG. 34. A schematic for a massive parallel way high density array ofmicro wells to perform the biochemical process. Each well can hold adifferent DNA template and nanopore device.

DETAILED DESCRIPTION OF THE INVENTION

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, with a DNA        polymerase and at least four deoxyribonucleotide polyphosphate        (dNPP) analogues under conditions permitting the DNA polymerase        to catalyze incorporation of one of the dNPP analogues into the        primer if it is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each of the four dNPP analogues has the        structure:

-   -    wherein the base is adenine, guanine, cytosine, thymine or        uracil, or a derivative of each thereof, wherein R₁ is OH,        wherein R₂ is H, wherein X is O, NH, S or CH₂, wherein n is 1,        2, 3, or 4, wherein Z is O, S, or BH₃, and with the proviso        that (i) the type of base on each dNPP analogue is different        from the type of base on each of the other three dNPP analogues,        and (ii) either the value of n of each dNPP analogue is        different from the value of n of each of the other three dNPP        analogues, or the value of n of each of the four dNPP analogues        is the same and the type of tag on each dNPP analogue is        different from the type of tag on each of the other three dNPP        analogues,    -    wherein incorporation of the dNPP analogue results in release        of a polyphosphate having the tag attached thereto; and    -   (b) determining which dNPP analogue has been incorporated into        the primer to form a DNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded DNA complementary to        the incorporated dNPP analogue; and    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded DNA being sequenced, wherein in        each iteration of step (a) the dNPP analogue is incorporated        into the DNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product,    -   thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, a DNA        polymerase and a deoxyribonucleotide polyphosphate (dNPP)        analogue under conditions permitting the DNA polymerase to        catalyze incorporation of the dNPP analogue into the primer if        it is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein the dNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, or a derivative of each thereof, wherein R₁ is —OH,        —O—CH₂N₃ or —O-2-nitrobenzyl, wherein R₂ is H, wherein X is O,        NH, S or CH₂, wherein n is 1, 2, 3, or 4, wherein Z is O, S, or        BH₃,    -    and wherein if the dNPP analogue is not incorporated,        iteratively repeating the contacting with a different dNPP        analogue until a dNPP analogue is incorporated, with the proviso        that (1) the type of base on each dNPP analogue is different        from the type of base on each of the other dNPP analogues,        and (2) either the value of n of each dNPP analogue is different        from the value of n of each of the other three dNPP analogues,        or the value of n of each of the four dNPP analogues is the same        and the type of tag on each dNPP analogue is different from the        type of tag on each of the other three dNPP analogues,    -    wherein incorporation of a dNPP analogue results in release of        a polyphosphate having the tag attached thereto;    -   (b) determining which dNPP analogue has been incorporated into        the primer to form a DNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded DNA complementary to        the incorporated dNPP analogue;    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded DNA being sequenced, wherein in        each iteration of step (a) the dNPP analogue is incorporated        into the DNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product,    -   thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        DNA has a primer hybridized to a portion thereof, with a DNA        polymerase and at least four deoxyribonucleotide polyphosphate        (dNPP) analogues under conditions permitting the DNA polymerase        to catalyze incorporation of one of the dNPP analogues into the        primer if it is complementary to the nucleotide residue of the        single-stranded DNA which is immediately 5′ to a nucleotide        residue of the single-stranded DNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a DNA extension        product, wherein each of the four dNPP analogues has a structure        chosen from the following:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, or a derivative of each thereof, wherein Y is a tag,        wherein R₁, if present, is OH, wherein R₂, if present, is H,        wherein X is a cleavable linker, wherein Z is O, S or BH₃,        wherein n is 1, 2, 3, or 4, wherein A is O, S, CH₂, CHF, CFF, or        NH, and with the proviso that (i) the type of base on each dNPP        analogue is different from the type of base on each of the other        three dNPP analogues, and (ii) the type of tag on each dNPP        analogue is different from the type of tag on each of the other        three dNPP analogues;    -   (b) cleaving the tag from the dNPP analogue incorporated in step        (a); and    -   (c) determining which dNPP analogue was incorporated in step (a)        by applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from tag cleaved        off in step (b) translocating through the nanopore, wherein the        electronic change is different for each different type of tag,        thereby identifying the nucleotide residue in the        single-stranded DNA complementary to the incorporated dNPP        analogue; and    -   (d) iteratively performing steps (a), (b) and (c) for each        nucleotide residue of the single-stranded DNA being sequenced,        wherein in each iteration of step (a) the dNPP analogue is        incorporated into the DNA extension product resulting from the        previous iteration of step (a) if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,    -   thereby determining the nucleotide sequence of the        single-stranded DNA.

A method for determining the nucleotide sequence of a single-strandedDNA comprising:

-   -   (a) contacting the single-stranded DNA, wherein the        single-stranded DNA is in an electrolyte solution in contact        with a nanopore in a membrane, wherein the single-stranded DNA        has a primer hybridized to a portion thereof, a DNA polymerase        and a deoxyribonucleotide polyphosphate (dNPP) analogue under        conditions permitting the DNA polymerase to catalyze        incorporation of the dNPP analogue into the primer if it is        complementary to the nucleotide residue of the single-stranded        DNA which is immediately 5′ to a nucleotide residue of the        single-stranded DNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a DNA extension product,        wherein the dNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, or derivative of each thereof, wherein Y is a tag, and        wherein R₁ if present is OH, —OCH₂N₃ or —O-2-nitrobenzyl, R₂ if        present is H, wherein X is a cleavable linker, wherein Z is O, S        or BH₃, wherein n is 1, 2, 3, or 4, wherein A is 0, S, CH₂, CHF,        CFF, or NH,    -    and if the dNPP analogue is not incorporated, iteratively        repeating the contacting with a different dNPP analogue until a        dNPP analogue is incorporated, with the proviso that (1) the        type of base on each dNPP analogue is different from the type of        base on each other dNPP analogue, and (2) the type of tag on        each dNPP analogue is different from the type of tag on each        other dNPP analogue, wherein incorporation of a dNPP analogue        results in release of a polyphosphate having the tag attached        thereto;    -   (b) cleaving the tag from the dNPP analogue incorporated in step        (a); and    -   (c) determining which dNPP analogue was incorporated in step (a)        to form a DNA extension product by applying a voltage across the        membrane and measuring an electronic change across the nanopore        resulting from the tag cleaved off in step (b) translocating        through the nanopore, wherein the electronic change is different        for each type of tag, thereby identifying the nucleotide residue        in the single-stranded DNA complementary to the incorporated        dNPP analogue;    -   (d) iteratively performing steps (a) through (c) for each        nucleotide residue of the single-stranded DNA being sequenced,        wherein in each iteration of step (a) the dNPP analogue is        incorporated into the DNA extension product resulting from the        previous iteration of step (a) if it is complementary to the        nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the DNA        extension product,    -   thereby determining the nucleotide sequence of the        single-stranded DNA.

In an embodiment of the methods the tag is ethylene glycol, an aminoacid, a carbohydrate, a dye, a mononucleotide, a dinucleotide, atrinucleotide, a tetranucleotide, a pentanucleotide or a hexanucleotide,a fluorescent dyes, a chemiluminescent compound, an amino acid, apeptide, a carbohydrate, a nucleotide monophosphate, a nucleotidediphosphate, an aliphatic acid or an aromatic acid or an alcohol or athiol with unsubstituted or substituted with one or more halogens, acyano group, a nitro group, an alkyl group, an alkenyl group, an alkynylgroup, an azido group.

In an embodiment of the methods the base is selected from the groupconsisting of adenine, guanine, cytosine, thymine, 7-deazaguanine,7-deazaadenine or 5-methylcytosine.

In an embodiment the methods further comprise a washing step after eachiteration of step (b) to remove unincorporated dNPP analogues fromcontact with the single-stranded DNA.

In an embodiment the methods further comprise a washing step after eachiteration of step (c) to remove unincorporated dNPP analogues fromcontact with the single-stranded DNA.

In an embodiment the methods further comprise wherein thesingle-stranded DNA, electrolyte solution and nanopore in the membraneare located within a single container.

In an embodiment of the methods wherein R₁ is —O—CH₂N₃, the methodsoptionally further comprise treating the incorporated dNPP analogue soas to remove the —CH₂N₃ and result in an OH group attached to the 3′position thereby permitting incorporation of a further dNPP analogue.

In an embodiment of the methods wherein R₁ is —O-2-nitrobenzyl, themethods optionally further comprise treating the incorporated nucleotideanalogue so as to remove the −2-nitrobenzyl and result in an OH groupattached to the 3′ position thereby permitting incorporation of afurther dNPP analogue.

In an embodiment of the methods the dNPP analogues have the followingstructures:

wherein R₁ is OH, wherein R₂ is H or OH, wherein Z is O, S, or BH₃, andwherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine.

In an embodiment of the methods the tag is a mononucleotide, adinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide or ahexanucleotide and wherein the base of the mononucleotide, thedinucleotide, the trinucleotide, the tetranucleotide, thepentanucleotide or the hexanucleotide is the same type of base as thebase of the dNPP analogue.

In an embodiment of the methods the tag is chosen from the following:

wherein in each structure n is, independently, 1, 2, 3 or 4, and m is,independently, an integer from 0 to 100, and wherein when m is 0 theterminal phosphate of the dNPP is bonded directly to the 3′ O atom ofthe nucleoside shown on the left hand side of the structure, and whereinthe value of n is different for each type of base.

In an embodiment of the methods m is an integer from 0 to 50. In anembodiment of the methods m is an integer from 0 to 10.

In an embodiment of the methods the dNPP analogue has the structure:

wherein R is a substituted or unsubstituted hydrocarbyl, up to 3000daltons, and wherein the base is adenine, guanine, cytosine, thymine,uracil, a 7-deazapurine or a 5-methylpyrimidine.

In an embodiment of the methods the dNPP analogue has the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine.

In an embodiment of the methods the dNPP analogue has the structure:

In an embodiment of the methods the dNPP analogue has the structure:

wherein m is an integer from 1-50, and wherein the base is adenine,guanine, cytosine, thymine, uracil, a 7-deazapurine or a5-methylpyrimidine.

In an embodiment of the methods the electronic change is a change incurrent amplitude.

In an embodiment of the methods the electronic change is a change inconductance of the nanopore.

In an embodiment of the methods the nanopore is biological. In anembodiment of the methods the nanopore is proteinaceous. In anembodiment of the methods the nanopore comprises alpha hemolysin. In anembodiment of the methods the nanopore is graphene. In an embodiment ofthe methods the nanopore is a solid-state nanopore. In an embodiment ofthe methods the nanopore is in a solid-state membrane.

In an embodiment of the methods the single stranded DNA, the primer, orthe DNA polymerase is attached to a solid surface.

In another embodiment of the methods the nanopore is part of an array ofnanopores.

A process for producing a nucleotide triphosphate analogue, wherein thenucleotide triphosphate analogue differs from a nucleotide triphosphateby having a tag attached to the terminal phosphate thereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        dicyclohexylcarbodiimide/dimethylformamide under conditions        permitting production of a cyclic trimetaphosphate;    -   b) contacting the product resulting from step a) with a tag        having a hydroxyl or amino group attached thereto under        conditions permitting nucleophilic opening of the cyclic        trimetaphosphate so as to bond the tag to a terminal phosphate        thereby forming the nucleotide triphosphate analogue.

A process for producing a nucleotide triphosphate analogue, wherein thenucleotide triphosphate analogue differs from a nucleotide triphosphateby having a tag attached to the terminal phosphate thereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        dicyclohexylcarbodiimide/dimethylformamide under conditions        permitting production of a cyclic trimetaphosphate;    -   b) contacting the product resulting from step a) with a        nucleophile so as to form an —OH or —NH₂ functionalized        compound;    -   c) reacting the product of step b) with a tag having a —COR        group attached thereto under conditions permitting the tag to        bond indirectly to a terminal phosphate thereby forming the        nucleotide triphosphate analogue.

In an embodiment of the instant process the nucleophile is H₂N—R—OH,H₂N—R—NH₂, R'S—R—OH, R'S—R—NH₂, or

In an embodiment the instant process comprises in step b) contacting theproduct resulting from step a) with a compound having the structure:

and then NH₄OH so as to form a compound having the structure:

and reacting the product of step b) with a tag having a —COR groupattached thereto under conditions permitting the tag to bond indirectlyto a terminal phosphate thereby forming the nucleotide triphosphateanalogue having the structure:

wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is adenine,guanine, cytosine, thymine, uracil, a 7-deazapurine or a5-methylpyrimidine.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a monophosphate group attached thereto under conditions        permitting formation of the nucleotide tetraphosphate analogue.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with phosphoric        acid under conditions permitting formation of a nucleotide        tetraphosphate;    -   c) contacting the nucleotide tetraphosphate with 1)        carbonyldiimidazole/dimethylformamide; 2) a nucleophile and        then 3) NH₄OH so as to form an —OH or —NH₂ functionalized        compound;    -   d) contacting the product of step c) with a tag having a —COR        group attached thereto under conditions permitting the tag to        bond indirectly to a terminal phosphate thereby forming the        nucleotide tetraphosphate analogue.

In an embodiment of the instant process the nucleophile is H₂N—R—OH,H₂N—R—NH₂, R'S—R—OH, R'S—R—NH₂, or

In an embodiment the instant process comprises in step b) contacting thenucleotide tetraphosphate with 1) carbonyldiimidazole/dimethylformamide;2) a compound having the structure:

and then 3) NH₄OH so as to form a compound having the structure:

and contacting the product of step b) with a tag having a —COR groupattached thereto under conditions permitting the tag to bond indirectlyto a terminal phosphate thereby forming the nucleotide triphosphateanalogue having the structure:

wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is adenine,guanine, cytosine, thymine, uracil, a 7-deazapurine or a5-methylpyrimidine.

A process for producing a nucleotide tetraphosphate analogue, whereinthe nucleotide tetraphosphate analogue differs from a nucleotidetetraphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -   b) contacting the product resulting from step a) with phosphoric        acid under conditions permitting formation of a nucleotide        tetraphosphate;    -   c) contacting the nucleotide tetraphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form a        compound having the structure:

-   -   wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine.

A process for producing a nucleotide pentaphosphate analogue, whereinthe nucleotide pentaphosphate analogue differs from a nucleotidepentaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a pyrophosphate group attached thereto under conditions        permitting formation of the nucleotide pentaphosphate analogue.

A process for producing a nucleotide pentaphosphate analogue, whereinthe nucleotide pentaphosphate analogue differs from a nucleotidepentaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a        pyrophosphate group under conditions permitting formation of a        nucleotide pentaphosphate;    -   c) contacting the nucleotide pentaphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form the        nucleotide pentaphosphate analogue.

A process for producing a nucleotide hexaphosphate analogue, wherein thenucleotide hexaphosphate analogue differs from a nucleotidehexaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a tag        having a triphosphate group attached thereto under conditions        permitting formation of the nucleotide hexaphosphate analogue.

A process for producing a nucleotide hexaphosphate analogue, wherein thenucleotide hexaphosphate analogue differs from a nucleotidehexaphosphate by having a tag attached to the terminal phosphatethereof, comprising:

-   -   a) contacting a nucleotide triphosphate with        1,1′-carbonyldiimidazole/dimethylformamide under conditions        permitting formation of the following structure:

-   -    wherein R₁ is OH, wherein R₂ is H or OH, wherein the base is        adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or        a 5-methylpyrimidine;    -   b) contacting the product resulting from step a) with a        triphosphate group under conditions permitting formation of a        nucleotide hexaphosphate;    -   c) contacting the nucleotide hexaphosphate with        carbonyldiimidazole/dimethylformamide and a tag having a        hydroxyl or amino group attached thereto so as to form the        nucleotide hexaphosphate analogue.

A compound having the structure:

-   -   wherein the tag is ethylene glycol, an amino acid, a        carbohydrate, a dye, mononucleotide, dinucleotide,        trinucleotide, tetranucleotide, pentanucleotide or        hexanucleotide, wherein R₁ is OH, wherein R₂ is H or OH, wherein        X is O, NH, S or CH₂, wherein Z is O, S, or BH₃, wherein the        base is adenine, guanine, cytosine, thymine, uracil, a        7-deazapurine or a 5-methylpyrimidine, and wherein n is 1, 2, 3,        or 4.

In an embodiment R₂ is H. In an embodiment R₂ is OH.

A compound having the structure:

wherein in each structure n is, independently, 1, 2, 3 or 4, and m is,independently, an integer from 0 to 100, and wherein when m is 0 theterminal phosphate of the dNTP is bonded directly to the 3′ O atom ofthe nucleoside shown on the left hand side of the structure, wherein R₁is —OH, or —O—CH₂N₃, and R₂ is H or OH.

In an embodiment m is from 0 to 50. In an embodiment m is from 0 to 10.In an embodiment R₁ is —OH. In an embodiment R₂ is —H. In an embodimentR₂ is —OH.

A compound having the structure:

wherein m an integer from 0 to 100, and wherein the compound comprises asingle type of base, and wherein the base is adenine, guanine, cytosine,uracil or thymine or a derivative thereof of each.

In an embodiment m is from 0 to 50. In an embodiment m is from 0 to 10.

In an embodiment the compound has the structure:

-   -   wherein m is an integer from 0 to 100.

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine.

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine, and R is a substituted orunsubstituted hydrocarbyl, up to 3000 daltons.

A compound having the structure:

A compound having the structure:

wherein the base is adenine, guanine, cytosine, thymine, uracil, a7-deazapurine or a 5-methylpyrimidine, and m is an integer from 1-50.

A compound having the structure:

wherein n is 1 or 2 and the base is adenine, guanine, cytosine, thymine,uracil, a 7-deazapurine or a 5-methylpyrimidine.

A compound having the structure:

wherein R₁ is —OH, or —O—CH₂N₃, and R₂ is H or OH.

A method for determining the nucleotide sequence of a single-strandedRNA comprising:

-   -   (a) contacting the single-stranded RNA, wherein the        single-stranded RNA is in an electrolyte solution in contact        with a nanopore in a membrane, wherein the single-stranded RNA        has a primer hybridized to a portion thereof, with a RNA        polymerase and at least four ribonucleotide polyphosphate (rNPP)        analogues under conditions permitting the RNA polymerase to        catalyze incorporation of one of the rNPP analogues into the        primer if it is complementary to the nucleotide residue of the        single-stranded RNA which is immediately 5′ to a nucleotide        residue of the single-stranded RNA hybridized to the 3′ terminal        nucleotide residue of the primer, so as to form a RNA extension        product, wherein each of the four rNPP analogues has the        structure:

-   -    wherein the base is adenine, guanine, cytosine, thymine or        uracil, or a derivative thereof of each, wherein R₁ is OH,        wherein R₂ is OH, wherein X is O, NH, S or CH₂, wherein n is 1,        2, 3, or 4, wherein Z is O, S, or BH₃, and with the proviso        that (i) the type of base on each rNPP analogue is different        from the type of base on each of the other three rNPP analogues,        and (ii) either the value of n of each rNPP analogue is        different from the value of n of each of the other three rNPP        analogues, or the value of n of each of the four rNPP analogues        is the same and the type of tag on each rNPP analogue is        different from the type of tag on each of the other three rNPP        analogues,    -    wherein incorporation of the rNPP analogue results in release        of a polyphosphate having the tag attached thereto; and    -   (b) determining which rNPP analogue has been incorporated into        the primer to form a RNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or for each        different type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded RNA complementary to        the incorporated rNPP analogue; and    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded RNA being sequenced, wherein in        each iteration of step (a) the rNPP analogue is incorporated        into the RNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded RNA which is immediately 5′ to a        nucleotide residue of the single-stranded RNA hybridized to the        3′ terminal nucleotide residue of the RNA extension product,    -   thereby determining the nucleotide sequence of the        single-stranded RNA.

A method for determining the nucleotide sequence of a single-strandedRNA comprising:

-   -   (a) contacting the single-stranded RNA, wherein the        single-stranded RNA is in an electrolyte solution in contact        with a nanopore in a membrane and wherein the single-stranded        RNA has a primer hybridized to a portion thereof, a RNA        polymerase and a ribonucleotide polyphosphate (rNPP) analogue        under conditions permitting the RNA polymerase to catalyze        incorporation of the rNPP analogue into the primer if it is        complementary to the nucleotide residue of the single-stranded        RNA which is immediately 5′ to a nucleotide residue of the        single-stranded RNA hybridized to the 3′ terminal nucleotide        residue of the primer, so as to form a RNA extension product,        wherein the rNPP analogue has the structure:

-   -    wherein the base is adenine, guanine, cytosine, uracil or        thymine, wherein R₁ is —OH, —O—CH₂N₃ or —O-2-nitrobenzyl,        wherein R₂ is —OH, wherein X is O, NH, S or CH₂, wherein n is 1,        2, 3, or 4, wherein Z is O, S, or BH₃,    -    and wherein if the rNPP analogue is not incorporated,        iteratively repeating the contacting with a different rNPP        analogue until a rNPP analogue is incorporated, with the proviso        that (1) the type of base on each rNPP analogue is different        from the type of base on each of the other rNPP analogues,        and (2) either the value of n of each rNPP analogue is different        from the value of n of each of the other three rNPP analogues,        or the value of n of each of the four rNPP analogues is the same        and the type of tag on each rNPP analogue is different from the        type of tag on each of the other three rNPP analogues,    -    wherein incorporation of a rNPP analogue results in release of        a polyphosphate having the tag attached thereto;    -   (b) determining which rNPP analogue has been incorporated into        the primer to form a RNA extension product in step (a) by        applying a voltage across the membrane and measuring an        electronic change across the nanopore resulting from the        polyphosphate having the tag attached thereto generated in        step (a) translocating through the nanopore, wherein the        electronic change is different for each value of n, or different        for each type of tag, as appropriate, thereby identifying the        nucleotide residue in the single-stranded RNA complementary to        the incorporated dNPP analogue;    -   (c) iteratively performing steps (a) and (b) for each nucleotide        residue of the single-stranded RNA being sequenced, wherein in        each iteration of step (a) the rNPP analogue is incorporated        into the RNA extension product resulting from the previous        iteration of step (a) if it is complementary to the nucleotide        residue of the single-stranded RNA which is immediately 5′ to a        nucleotide residue of the single-stranded RNA hybridized to the        3′ terminal nucleotide residue of the RNA extension product,    -   thereby determining the nucleotide sequence of the        single-stranded RNA.

In an embodiment the dNPP analogue has the structure:

wherein n is 1 or 2 and the base is adenine, guanine, cytosine, thymine,uracil, a 7-deazapurine or a 5-methylpyrimidine.

In an embodiment the biological nanopore is integrated with CMOSelectronics. In another embodiment the solid-state nanopore isintegrated with CMOS electronics.

In an embodiment the attachment to the solid surface is viabiotin-streptavidin linkages. In another embodiment the DNA polymeraseis attached to the solid surface via gold surface modified with analkanethiol self-assembled monolayer functionalized with amino groups,wherein the amino groups are modified to NHS esters for attachment toamino groups on the DNA polymerase.

In one embodiment the dNPP analogue is a terminal-phosphate-taggednucleoside-polyphosphate. In a further embodiment each type of dNPPanalogue has a polyethylene glycol tag which differs in size from thepolyethylene glycol tags of each of the other three types of dNPPanalogues.

In one embodiment the tag has the structure as follows:

wherein W is an integer between 0 and 100.

In another embodiment the tag has the structure as follows:

wherein R is NH₂, OH, COOH, CHO, SH, or N₃, and W is an integer from 0to 100.

A composition comprising at least four deoxynucleotide polyphosphate(dNPP) analogues, each having a structure selected from the structuresset forth in claims 74 and 75, wherein each of the four dNPP analoguescomprises a type of base different from the type of base of the otherthree dNPP analogues.

In one embodiment, each of the four dNPP analogues has a polyethyleneglycol tag which is different in size from the polyethylene glycol tagsof each of the other three dNPP analogues.

In an embodiment net charge on the tagged nucleoside polyphosphate isneutral. In another embodiment the released tag has a positive charge.

In one embodiment, the method further comprising a step of treating withalkaline phosphatase after step b), wherein the alkaline phosphatasehydrolyzes free phosphate groups on the released tag-pyrophosphate.

In one embodiment multiple copies of the single-stranded DNA areimmobilized on a bead.

A “derivative” of adenine, guanine, cytosine, thymine or uracil, includea 7-deaza-purine and a 5-methyl pyrimidine. Examples include 7-deazaadenine, 7-deaza-guanine, and 5-methyl-cytosine.

The present invention also provides a compound having the structure ofany of the compounds set forth in the figures and/or schemes of thepresent application.

The present invention also provides a dNPP analogue comprising a taghaving the structure of any of the tags set forth in the figures and/orschemes of the present application.

In an embodiment, the tag is a hydrocarbyl, substituted orunsubstituted, such as an alkyl, alkenyl, alkynyl, and having a mass of3000 daltons or less.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in“C1-Cn alkyl” is defined to include groups having 1, 2, . . . , n−1 or ncarbons in a linear or branched arrangement. For example, a “C1-C5alkyl” is defined to include groups having 1, 2, 3, 4, or 5 carbons in alinear or branched arrangement, and specifically includes methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, and pentyl

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C2-C5 alkenyl” means an alkenyl radicalhaving 2, 3, 4, or 5, carbon atoms, and up to 1, 2, 3, or 4,carbon-carbon double bonds respectively. Alkenyl groups include ethenyl,propenyl, and butenyl.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C2-C5alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

The term “substituted” refers to a functional group as described abovesuch as an alkyl, or a hydrocarbyl, in which at least one bond to ahydrogen atom contained therein is replaced by a bond to non-hydrogen ornon-carbon atom, provided that normal valencies are maintained and thatthe substitution(s) result(s) in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Non-limiting examples of substituentsinclude the functional groups described above, and for example, N, e.g.so as to form —CN.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

In the compound structures depicted herein, hydrogen atoms, except onribose and deoxyribose sugars, are generally not shown. However, it isunderstood that sufficient hydrogen atoms exist on the representedcarbon atoms to satisfy the octet rule.

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

A—Adenine; C—Cytosine;

DNA—Deoxyribonucleic acid;

G—Guanine;

RNA—Ribonucleic acid;

T—Thymine; and U—Uracil.

dNPP—deoxyribonucleotide polyphosphaterNPP—ribonucleotide polyphosphate

“Nucleic acid” shall mean, unless otherwise specified, any nucleic acidmolecule, including, without limitation, DNA, RNA and hybrids thereof.In an embodiment the nucleic acid bases that form nucleic acid moleculescan be the bases A, C, G, T and U, as well as derivatives thereof.Derivatives of these bases are well known in the art, and areexemplified in PCR Systems, Reagents and Consumables (Perkin ElmerCatalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J.,USA).

A nucleotide polyphosphate, such as a deoxyribonucleotide polyphosphate(“dNPP”) or a ribonucleotide polyphosphate “(rNPP”), is a nucleotidecomprising multiple, i.e. three, four, five, six, or more phosphates ina linear fashion bonded to its 5′ sugar carbon atom. A nucleotidepolyphosphate analogue is an analogue of such a deoxyribonucleotidepolyphosphate or of such a ribonucleotide polyphosphate as definedherein, differing thereform by having a tag attached thereto at aspecified position. Such analogues are incorporable into a primer ornucleic acid extension strand, such as a DNA extension strand, bycontacting with an appropriate nucleic acid polymerase under theappropriate nucleic acid polymerization conditions known to those in theart.

In one embodiment the dNPP is a deoxynucleotide triphosphate.

As used herein a tetranucleotide, a pentanucleotide, or ahexanucleotide, encompasses 4, 5 or 6, respectively, nucleic acidmonomer residues joined by phosphodiester bonds, wherein the freeterminal residue can be a nucleotide or a nucleoside. In an embodiment,the free terminal residue is a nucleoside and the other residues arenucleotides.

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid may be affixed. Non-limiting examplesinclude chips, wells, beads, nanopore structures and columns. In anon-limiting embodiment the solid substrate can be present in asolution, including an aqueous electrolyte solution.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid (such as primer) based on the well-understoodprinciple of sequence complementarity. In an embodiment the othernucleic acid is a single-stranded nucleic acid. The propensity forhybridization between nucleic acids depends on the temperature and ionicstrength of their milieu, the length of the nucleic acids and the degreeof complementarity. The effect of these parameters on hybridization iswell known in the art (see Sambrook J, Fritsch E F, Maniatis T. 1989.Molecular cloning: a laboratory manual. Cold Spring Harbor LaboratoryPress, New York). As used herein, hybridization of a primer sequence, orof a DNA extension product, to another nucleic acid shall mean annealingsufficient such that the primer, or DNA extension product, respectively,is extendable by creation of a phosphodiester bond with an availablenucleotide or nucleotide analogue capable of forming a phosphodiesterbond, therewith.

As used herein, unless otherwise specified, a base which is “differentfrom” another base or a recited list of bases shall mean that the basehas a different structure than the other base or bases. For example, abase that is “different from” adenine, thymine, and cytosine wouldinclude a base that is guanine or a base that is uracil.

“Primer” as used herein (a primer sequence) is a short, usuallychemically synthesized oligonucleotide, of appropriate length, forexample about 18-24 bases, sufficient to hybridize to a target DNA (e.g.a single stranded DNA) and permit the addition of a nucleotide residuethereto, or oligonucleotide or polynucleotide synthesis therefrom, undersuitable conditions well-known in the art. In an embodiment the primeris a DNA primer, i.e. a primer consisting of, or largely consisting of,deoxyribonucleotide residues. The primers are designed to have asequence which is the reverse complement of a region of template/targetDNA to which the primer hybridizes. The addition of a nucleotide residueto the 3′ end of a primer by formation of a phosphodiester bond resultsin a DNA extension product. The addition of a nucleotide residue to the3′ end of the DNA extension product by formation of a phosphodiesterbond results in a further DNA extension product.

In an embodiment the single-stranded DNA, RNA, primer or probe is boundto a solid substrate via 1,3-dipolar azide-alkyne cycloadditionchemistry. In an embodiment the DNA, RNA, primer or probe is bound to asolid substrate via a polyethylene glycol molecule. In an embodiment theDNA, RNA, primer or probe is alkyne-labeled. In an embodiment the DNA,RNA, primer or probe is bound to a solid substrate via a polyethyleneglycol molecule and a solid substrate is azide-functionalized In anembodiment the DNA, RNA, primer or probe is immobilized on the solidsubstrate via an azido linkage, an alkynyl linkage, orbiotin-streptavidin interaction. Immobilization of nucleic acids isdescribed in Immobilization of DNA on Chips II, edited by ChristineWittmann (2005), Springer Verlag, Berlin, which is hereby incorporatedby reference. In an embodiment the DNA is single-stranded DNA. In anembodiment the RNA is single-stranded RNA.

In an embodiment the solid substrate is in the form of a chip, a bead, awell, a capillary tube, a slide, a wafer, a filter, a fiber, a porousmedia, a porous nanotube, or a column. This invention also provides theinstant method, wherein the solid substrate is a metal, gold, silver,quartz, silica, a plastic, polypropylene, a glass, or diamond. Thisinvention also provides the instant method, wherein the solid substrateis a porous non-metal substance to which is attached or impregnated ametal or combination of metals. The solid surface may be in differentforms including the non-limiting examples of a chip, a bead, a tube, amatrix, a nanotube. The solid surface may be made from materials commonfor DNA microarrays, including the non-limiting examples of glass ornylon. The solid surface, for example beads/micro-beads, may be in turnimmobilized to another solid surface such as a chip.

In an embodiment nucleic acid samples, DNA, RNA, primer or probe areseparated in discrete compartments, wells or depressions on a surface orin a container.

This invention also provides the instant method, wherein about 1000 orfewer copies of the nucleic acid sample, DNA, RNA, primer or probe, arebound to the solid surface. This invention also provides the instantinvention wherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of thenucleic acid sample, DNA, RNA, primer or probe are bound to the solidsurface.

In an embodiment the immobilized nucleic acid sample, DNA, RNA, primeror probe is immobilized at a high density. This invention also providesthe instant invention wherein over or up to 1×10⁷, 1×10⁸, 1×10⁹ copiesof the nucleic acid sample, DNA, RNA, primer or probe, are bound to thesolid substrate.

In an embodiment the DNA polymerase is 9° N polymerase or a variantthereof, E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase,Sequenase, Taq DNA polymerase or 9° N polymerase (exo-)A485L/Y409V.

In an embodiment of the methods or of the compositions described herein,the DNA is single-stranded. In an embodiment of the methods or of thecompositions described herein, the RNA is single-stranded, Phi29, orvariants thereof.

In an embodiment of the methods described for RNA sequencing, thepolymerase is an RNA polymerase, reverse transcriptase or appropriatepolymerase for RNA polymerization as known in the art.

The linkers may be photocleavable. In an embodiment UV light is used tophotochemically cleave the photochemically cleavable linkers andmoieties. In an embodiment, the photocleavable linker is a 2-nitrobenzylmoiety.

The —CH₂N₃ group can be treated with TCEP(tris(2-carboxyethyl)phosphine) so as to remove it from the 3′ O atom ofa dNPP analogue, or rNPP analogue, thereby creating a 3′ OH group.

Methods for production of cleavably capped and/or cleavably linkednucleotide analogues are disclosed in U.S. Pat. No. 6,664,079, which ishereby incorporated by reference.

A “nucleotide residue” is a single nucleotide in the state it existsafter being incorporated into, and thereby becoming a monomer of, apolynucleotide. Thus, a nucleotide residue is a nucleotide monomer of apolynucleotide, e.g. DNA, which is bound to an adjacent nucleotidemonomer of the polynucleotide through a phosphodiester bond at the 3′position of its sugar and is bound to a second adjacent nucleotidemonomer through its phosphate group, with the exceptions that (i) a 3′terminal nucleotide residue is only bound to one adjacent nucleotidemonomer of the polynucleotide by a phosphodiester bond from itsphosphate group, and (ii) a 5′ terminal nucleotide residue is only boundto one adjacent nucleotide monomer of the polynucleotide by aphosphodiester bond from the 3′ position of its sugar.

Because of well-understood base-pairing rules, determining the identity(of the base) of dNPP analogue (or rNPP analogue) incorporated into aprimer or DNA extension product (or RNA extension product) by measuringthe unique electrical signal of the tag translocating through thenanopore, and thereby the identity of the dNPP analogue (or rNPPanalogue) that was incorporated, permits identification of thecomplementary nucleotide residue in the single stranded polynucleotidethat the primer or DNA extension product (or RNA extension product) ishybridized to. Thus, if the dNPP analogue that was incorporatedcomprises an adenine, a thymine, a cytosine, or a guanine, then thecomplementary nucleotide residue in the single stranded DNA isidentified as a thymine, an adenine, a guanine or a cytosine,respectively. The purine adenine (A) pairs with the pyrimidine thymine(T). The pyrimidine cytosine (C) pairs with the purine guanine (G).Similarly, with regard to RNA, if the rNPP analogue that wasincorporated comprises an adenine, an uracil, a cytosine, or a guanine,then the complementary nucleotide residue in the single stranded RNA isidentified as an uracil, an adenine, a guanine or a cytosine,respectively.

Incorporation into an oligonucleotide or polynucleotide (such as aprimer or DNA extension strand) of a dNPP or rNPP analogue means theformation of a phosphodiester bond between the 3′ carbon atom of the 3′terminal nucleotide residue of the polynucleotide and the 5′ carbon atomof the dNPP analogue or rNPP analogue, respectively.

As used herein, unless otherwise specified, a base (e.g. of a nucleotidepolyphosphate analogue) which is different from the type of base of areferenced molecule, e.g. another nucleotide polyphosphate analogue,means that the base has a different chemical structure from theother/reference base or bases. For example, a base that is differentfrom adenine would include a base that is guanine, a base that isuracil, a base that is cytosine, and a base that is thymine. Forexample, a base that is different from adenine, thymine, and cytosinewould include a base that is guanine and a base that is uracil.

As used herein, unless otherwise specified, a tag (e.g. of a nucleotidepolyphosphate analogue) which is different from the type of tag of areferenced molecule, e.g. another nucleotide polyphosphate analogue,means that the tag has a different chemical structure from the chemicalstructure of the other/referenced tag or tags.

“Nanopore” includes, for example, a structure comprising (a) a first anda second compartment separated by a physical barrier, which barrier hasat least one pore with a diameter, for example, of from about 1 to 10nm, and (b) a means for applying an electric field across the barrier sothat a charged molecule such as DNA, nucleotide, nucleotide analogue, ortag, can pass from the first compartment through the pore to the secondcompartment. The nanopore ideally further comprises a means formeasuring the electronic signature of a molecule passing through itsbarrier. The nanopore barrier may be synthetic or naturally occurring inpart. Barriers can include, for example, lipid bilayers having thereinα-hemolysin, oligomeric protein channels such as porins, and syntheticpeptides and the like. Barriers can also include inorganic plates havingone or more holes of a suitable size. Herein “nanopore”, “nanoporebarrier” and the “pore” in the nanopore barrier are sometimes usedequivalently.

Nanopore devices are known in the art and nanopores and methodsemploying them are disclosed in U.S. Pat. Nos. 7,005,264 B2; 7,846,738;6,617,113; 6,746,594; 6,673,615; 6,627,067; 6,464,842; 6,362,002;6,267,872; 6,015,714; 5,795,782; and U.S. Publication Nos. 2004/0121525,2003/0104428, and 2003/0104428, each of which are hereby incorporated byreference in their entirety.

In an embodiment of the molecules and the methods disclosed herein thetag is attached to the remainder of the molecule by a chemical linkerwhich is cleavable.

In an embodiment the nanpore is in a solid-state membrane. In anembodiment the membrane is a silicon nitride membrane. In an embodimentthe nanopore is a biopore. In an embodiment the pore is proteinaceous.In an embodiment the pore is an alpha-hemolysin pore. In an embodimentthe pore is a graphene pore.

In an embodiment the DNA, RNA or single stranded nucleic acid is locatedon one side of the membrane in which the nanopore is located and themembrane is located in a conducting electrolyte solution.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention.

All combinations of the various elements described herein are within thescope of the invention. All sub-combinations of the various elementsdescribed herein are also within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS AND DISCUSSIONS

The invention disclosed herein pertains to modified nucleotides forsingle molecule analysis of DNA (or RNA, mutatis mutandis) usingnanopores. Modifications can be made at various positions of anucleotide, i.e. the terminal phosphate, the base, and/or the 2′, or3′-OH to form a nucleotide analogue. After a polymerase extensionreaction on a template-primer complex, the released tag-attachedpyrophosphate passes through a nanopore and the resulting currentblockage is monitored to determine the nucleotide base added. If themodification or tag is at the base moiety, or the 2′/3′-OH of the sugarmoiety of the nucleotide, then after incorporation by DNA/RNApolymerase, the linker-tag is cleaved from the base/sugar by chemical orphotochemical means and released linker-tag passes through a nanopore toidentify the added nucleotide.

Nucleoside-5′-polyphosphates carrying different number of phosphategroups as linkers and modified with tags attached to the terminalphosphate of the nucleotides are designed and synthesized. Afterincorporation by DNA/RNA polymerase in a template-primer extensionreaction, the released tag-attached polyphosphate (di-, tri-, tetra-,penta-, etc.) can be detected using a nanopore to produce sequence data.Optionally, the released tag-polyphosphates can also be treated withalkaline phosphatase to provide free tags. Using four different tagswhich are distinct and specific for each nucleotide base, the sequenceof the template DNA or RNA can be determined.

Nucleotides carrying different number of phosphate groups or tags forthe synthesis of modified nucleotides, which are efficient substrates inpolymerase reactions, are provided. The released tag-attachedpolyphosphate is detected using a nanopore to determine conditions fordesign and modification of the nucleotides to achieve distinct blockadesignals.

Also provided are nucleotides carrying linker-tag attached at thenucleotide base moiety, and/or the 2′/3′-OH of the sugar moiety, for DNApolymerase reaction to generate linker-tag labeled single base DNAextension product. These nucleotides are good substrates for commonlyused DNA/RNA polymerases. The linker-tag attached at the extended DNAproduct is cleaved by chemical or photochemical means to generate theprimer ready for further extension using the modified nucleotides. Thereleased linker-tag is passed through nanopore and identified based onthe difference in size, shape, and charge on the tag to produce sequencedata.

As disclosed herein, these molecular tools facilitate single moleculesequencing using nanopore at single base resolution.

Here are disclosed several improvements to the nanopore approach: 1) toachieve accurate and obvious discrimination of the four bases (A, C, Gand T) that make up the nucleic acid molecules; 2) to enhance anddifferentiate the strength of the detection signals; 3) to develop aneffective method for discerning and processing the electronic blockadesignals generated; 4) to control the translocation rate of nucleic acidsthrough the pore, such as slowing down the movement of tags to improvethe ability of base-to-base discrimination; and 5) to design and makenew and more effective synthetic nanopores for differentiating the fourdifferent nucleotides in DNA.

The structures of four nucleotides are shown in FIG. 2. A and G arepurines, while C and T are pyrimidines. The overall molecular size of Aand G is very similar, while the size of C and T is similar. Nanoporeshave been shown to be able to differentiate between purines andpyrimidines [Akeson et al. 1999 and Meller et al. 2000], but not be ableto distinguish between individual purines, A and G, or betweenindividual pyrimidines, C and T.

Previous studies have shown modifications ofnucleoside-5′-triphosphates, including introducing more phosphate groupsto produce tetra-, penta-, or hexa-phosphates, introducing dye directlyto the terminal phosphate, or attaching a linker between the terminalphosphate and the dye [Kumar et al., 2006 and 2008]. Tetra- andpenta-phosphates are better DNA polymerase substrates, and dye-labeledhexa-phosphate nucleotides have been developed [Kumar et al. 2005; Soodet al. 2005; Eid et al. 2009].

Nucleotide analogues which are designed to enhance discrimination ofeach nucleotide by modification of the nucleotides at the terminalphosphate moiety are disclosed herein. Nucleoside-5′-polyphosphates aresynthesized and different tags (such as, different length/masspoly(ethylene glycol)s (PEGs), amino acids, carbohydrates,oligonucleotides, dyes or organic/inorganic molecules) are attached tothe terminal phosphate group. After polymerase extension reactions,tag-attached polyphosphate moieties are generated (FIG. 3) and differentsignal specific to each base is produced when the tag-attachedpolyphosphate moieties pass through the nanopore. These modificationsenlarge the discrimination of the bases by nanopore due to the increasedsize, mass or charge differences of released tagged-polyphosphate unitsbetween the four nucleotides (A, G, C and T).

The DNA translocation rate through the nanopore is reduced due to thebulkiness of the released tag-attached polyphosphates, although thetranslocation rate of the tags through the nanopore does not need to bereduced as long as the tags can be differentiated. Thus, the accuracyand reliability required for the base-to-base sequencing becomesachievable. Other analytical parameters in nanopore sequencing, such asconcentration of the polynucleotide, magnitude of the applied voltage,temperature and pH value of the solution, are optimized in order to getthe most accurate and reliable results for the detection and analysis ofDNA chain.

Single-molecule approaches to sequencing allow for the possibility ofderiving haplotypes for genetic studies and permitting direct sequencingof mRNAs. Among the potential single-molecule approaches for decodingthe sequence of DNA or RNA molecules is the use of biological orsynthetic nanopores as detectors of the individual DNA bases.

Existing sequencing-by-synthesis (SBS) approach uses cleavablefluorescent nucleotide reversible terminators (CF-NRTs) [Guo et al.2010]. SBS method is based on the ability to pause after each nucleotideaddition during the polymerase reaction and the use of specificfluorophores to discriminate among the bases. However, a majorlimitation of SBS for single molecule sequencing is the requirement forexpensive fluorescence detectors and rapid imaging software. The methodand process disclosed herein harness the advantages of SBS, especiallyits high accuracy, with the speed and sensitivity of the nanopore as anionic current impedance detector.

While much research has gone into threading DNA through nanopores, withthe hope of discriminating each base as it passes through due to itsvariable effect on the ion current, this has been very hard to achieve,both due to the speed of transmission and the effect of surroundingbases which may contribute their own effects on ions and counter ionspassing through the pores [Timp et al. 2010]. The use of cyclodextrinsor other ring-shaped structures in the lumen of protein pores helpprovide a ratcheting mechanism to slow down transit time [Astier et al.2006], but the ability to absolutely recognize each base for sequencingas it passes remains a challenge. An alternative strategy which usesexonuclease to allow one nucleotide at a time to traverse the pore hasled to single base discrimination [Clarke et al. 2009]. However, thereis still difficulty in controlling the reaction time of the exonucleasefor different lengths of DNA and nucleotide and the speed at which thereleased ions arrive at the pore with this approach.

Polymerase reaction itself displays high processivity and stable ratesof base incorporation. Indeed, polymerase reactions have been used tocontrol the movement of DNA strands through nanopores for direct basediscrimination [Benner et al. 2007, Cockroft et al. 2008, Hurt et al.2009]. During the polymerase reaction, there is release of apyrophosphate (PPi) moiety. Therefore, if one attaches a different tagto the triphosphate for each of the four nucleotides, these could bediscriminated as they are released and pass through an appropriatenanopore for DNA sequence determination. These relatively smallpyrophosphate analogs, or equivalent molecules with additionalpositively charged groups, can reach the pore extremely rapidly. Therate of nucleotide incorporation by polymerases is approximately 1000nucleotides per second, i.e. a millisecond per base addition, while thetransport rate through the nanopore is 1 molecule per microsecond. Thus,with proper fluidics and engineering, there are no de-phasing issues tosequence DNA with our approach, nor are there difficulties with thedecoding of homopolymer stretches. It has been shown that one candiscriminate among a wide size range of polyethylene glycols differingby as little as one or two carbon units by the effect they have onblocking currents in nanopores [Reiner et al. 2010, Robertson et al.2007], a resolution essentially equivalent to that obtained by a massspectrometer. Therefore, as described below, different length PEG chainsare attached to the terminal phosphate of dATP, dCTP, dGTP and dTTP. Aseach nucleotide is incorporated during the polymerase reaction, aspecifically tagged phosphate group is released into the nanopore,yielding a distinct current blockade signal to indicate which nucleotideis incorporated. The speed of sequencing is extremely fast, limited onlyby the rate of the polymerase reaction. As an alternative approach fortagging the nucleotides, we also utilize different phosphate chainlengths (e.g., tri-, tetra-, and penta-phosphates).

Additionally, we also use solid-state nanopores which have advantages interms of better control over and flexibility of fabrication, thus ensurerapid vectorial transport of tagged polyphosphates but not thenucleotide precursors or the DNA toward and through the nanopores ornanochannels. To achieve this, two important design features areincorporated. First, the precursors (tagged nucleotide polyphosphates)are synthesized with an overall neutral charge, while the cleaved taggedphosphates have an overall positive charge. By utilizing a current thatattracts positive ions, the nanopores only need to discriminate the fouralternative released tagged molecules. Differential charge on precursorsand products are achieved by incorporate into the tags a number oflysines or arginines (positively charged) exactly balancing the numberof phosphates (negatively charged). After incorporation of theα-phosphate into the growing primer, there is one more lysine thanphosphate in the released product. Optionally, alkaline phosphatase canbe used to cleave off all the phosphates to produce a PEG tag with astronger positive charge. Second, to assure that the released phosphatesmove immediately through the nearest pore, the DNA polymerase isimmobilized to the inlet of the pore, for example via abiotin-streptavidin linkage. As the DNA chain threads through thepolymerase, the released tagged products only have to diffuse the sameshort distance to reach the nanopore.

It is also important to recognize the advantages of the bioelectronictransduction mechanism over optical approaches. For single-moleculeoptical transduction techniques, the signal from a single-fluorophore istypically <2500 photons/sec (corresponding to detected current levels onthe order of 50 fA) at high short noise levels, requiring complex opticsto try to collect every photon emitted, making scaling of the platformsto higher densities difficult. Synthesis reactions must be slowed to 1Hz to allow sufficient integration times for these weak, noisy opticalsignals. The challenges to optical techniques have opened up thepossibility for bioelectronic detection approaches, which havesignificantly higher signal levels (typically more than three orders ofmagnitude higher), allowing for the possibility for high-bandwidthdetection with the appropriate co-design of transducer, detector, andamplifier. Signal levels for nanopores can be as high as 100 pA fromalpha-hemolysin [Kasianowicz et al. 1996], 300 pA for MspA [Derringtonet al. 2010], and upwards of 4 nA from solid-state nanopores [Wanunu etal. 2010].

Significant effort has been directed toward the development of nanoporetechnology as a bioelectronic transduction mechanism [Benner et al.2007, Deamer et al. 2002, Kasianowicz et al. 1996, Branton 2008, Brantonet al. 2008, Chen 2004, Gershow et al. 2007, Nealy 2007, Matysiak et al.2006]. Two essential attributes of this electronic sensor give itsingle-molecule sensitivity. The first is the very localized (nanoscale)geometry of charge sensitivity in the pore itself. The diameter of apore might be 2-3 nm, and due to electrolyte charge screening themeasured current is highly insensitive to charge sources more than a fewnanometers from the pore. Second, the nanopore sensor provides a gainthrough the effect the comparatively slow-moving charge a biopolymer hason a nearby concentration of higher-mobility salt ions. Nanopores,however, are extremely limited by the relatively short time biomoleculesspend in the charge-sensitive region of the pore. This is directlyaddressed by the use of tags, which can be optimized to produce highsignal levels and longer translocation events. At the same time, CMOSco-integration of these pores is exploited to dramatically improve thenoise-limited bandwidths for detection in a nanopore device. Bothsolid-state and biological pores are supported by this platform. Thissolid-state integration, along with associated microfluidics, alsouniquely enables the scale-up of this design to large arrays withintegrated electronics for detection.

Example 1 I. Design and Synthesis of Modified Nucleotides

Effect of bulkiness of the tagged-polyphosphate on electronic blockadesignals generated by a nanopore is determined using variousphosphate-linked-nucleotides with different size tags or groups attachedto the terminal phosphate of the nucleotide. Structures of fourphosphate-tagged nucleoside-5′-polyphosphates are shown in FIG. 4.First, a series of nucleoside-5′-tri-, tetra-, penta-, andhexa-phosphates is synthesized. In these nucleotides, the terminalphosphate is attached with a linker through which different tags, e.g.different length and mass ethylene glycols or other molecules whichincreases the bulkiness or charge of the released polyphosphate, areattached. These nucleotides are tested with nanopore to determine whichtags or bulky groups attached to the terminal phosphate correlate tomore dramatic difference in electronic blockade signal between thedifferent bases.

1) Terminal Phosphate-Modified Nucleoside-Polyphosphates

a. Terminal Phosphate-Tagged Nucleoside-5′-triphosphates

As shown in FIG. 5, terminal phosphatetagged-nucleoside-5′-triphosphates can be synthesized by reacting thecorresponding dNTP with DCC/DMF to give cyclic trimetaphosphate whichcan be opened with appropriate nucleophiles to give tag or linkerattached nucleoside-5′-triphosphate. This can be used in atemplate-primer extension reaction and the released tag-attachedpyrophosphate can be read using nanopore. Alternatively, the linkerattached to the phosphate can be reacted with tag-NHS ester to providealternate tag-attached nucleoside-5′-triphosphate.

b. Terminal Phosphate-Tagged Nucleoside-5′-tetraphosphates

For the synthesis of terminal phosphate taggednucleoside-5′-tetraphosphates, the corresponding triphosphate is firstreacted with CDI in DMF to activate the terminal phosphate group whichis then reacted with phosphoric acid or tag-monophosphate to give thetetraphosphate (FIG. 6). The terminal phosphate on the tetraphosphatecan be further activated with CDI followed by reaction with appropriatenucleophiles to provide a linker attached tetraphosphate which canfurther be used to attach tags of different mass, length or bulk, suchas m-dPEG-NHS ester, also shown in FIG. 6.

c. Terminal Phosphate-Tagged Nucleoside-5′-penta- and hexaphosphates

Synthesis of terminal phosphate tagged nucleoside-5′-penta- andhexaphosphates follows the same principle as shown in FIG. 7. They canbe prepared either from activated triphosphates or the tetraphosphatesby reacting with phosphoric acid, pyrophosphate or tag-attachedphosphates. Alternatively, a linker can be attached to penta- orhexa-phosphate followed by reaction with activated NHS esters.

d. Oligo-Tag Attached Nucleoside-Polyphosphates

There are a number of issues with current approach to nanoporesequencing such as recognition of the bases as they pass through thenanopore and the speed or rate of transport to allow recognition of thenucleobase be registered. DNA passes through a α-hemolysin nanopore at arate of 1-5 μs, which is too fast to record for single moleculesequencing experiments. Some progress has been made to overcome theseissues by a variety of protein engineering strategies including the useof molecular brakes (short covalently attached oligonucleotides)[Bayley, H. 2006].

As disclosed herein, short oligonucleotides can be attached to theterminal-phosphate of a nucleoside polyphosphate by reaction of theactivated terminal phosphate with the 3′-OH or the 5′-OH of theoligonucleotide. Alternatively, the 3′- or 5′-phosphate of theoligonucleotide can be activated with CDI or Imidazole/DCC and reactedwith nucleoside-5′-polyphosphates. Structures of oligo-attachednucleoside phosphates (oligo-3′ to 5′-phosphate; oligo-5′ to5′-phosphate) are shown in FIGS. 8(a) and 8(b), respectively. Thepolymerase reaction by-product which is monitored by passing through thenanopore is shown in FIG. 8(c).

The rate of migration through the nanopore of the polymerase reactionby-product can be controlled by attaching oligonucleotides of differentlength to different nucleoside-5′-polyphosphates. For example, ifnucleoside dA has 1 or 2 oligo-dA units attached, dT may have 3 oligo-dTunits, dC may have 4 oligo-dC units, and dG may have 5 oligo-dG units.Different combinations of the number of oligos for each nucleotide couldbe used to control the transport and retention time in a nanopore.

The transport and retention time in a nanopore also can be controlled byadding different number of phosphate groups to the nucleotides. Thus thecharge and mass can vary for each nucleotide polyphosphate.

Examples of Linker Tag Structure

Specific examples of reactive groups on the terminal phosphates or thenucleoside base moiety and groups with which groups can react areprovided in Table 1. The reactive groups with which they can react canbe present either on the linker or on the tag.

TABLE 1 Possible Reactive Substituents and Functional Groups ReactiveTherewith Reactive Groups Functional Groups Succinimidyl esters Primaryamino, secondary amino Anhydrides, acid halides Amino and Hydroxylgroups Carboxyl Amino, Hydroxy, Thiols Aldehyde, Isothiocyanate &Isocyanates Amino groups Vinyl sulphone & Dichlorotriazine Amino groupsHaloacetamides Thiols, Imidazoles Maleimides Thiols, Hydroxy, AminoThiols Thiols, Maleimide, Haloacetamide Phosphoramidites, Activated P.Hydroxy, Amino, Thiol groups Azido Alkyne

Tags which can be detected by nanopore are included herewith but by nomeans are they limited to these group of compounds. One skilled in theart may change the functional group(s) to come up with a suitable tag.

The tags include aliphatic, aromatic, aryl, heteroaryl compounds withone or more 4-8 membered rings and may optionally be substituted withhalo, hydroxy, amino, nitro, alkoxy, cyano, alkyl, aryl, heteroaryl,acid, aldehyde, Azido, alkenyl, alkynyl, or other groups. Theseincludes, polyethylene glycols (PEGs), carbohydrates, aminoacids,peptides, fluorescent, fluorogenic (non-fluorescent but becomefluorescent after removal of protecting group) chromogenic (colorlessbut become colored after removal of protecting group) dyes,chemiluminiscent compounds, nucleosides, nucleoside-mono, di orpolyphosphates, oligonucleotides, aryl, heteroaryl or aliphaticcompounds. Some examples are given in FIG. 16.

Structure of PEG-phosphate-labeled nucleotides and some examples ofpossible PEGs with different reactive groups to react with functionalgroups are exemplified in FIG. 17.

Some other examples of the dyes or compounds which can be used to attachto the terminal phosphate or the base moiety of the nucleotides areprovided here. By no means, these are the only compounds which can beused. These are listed here as examples and one skilled in the art caneasily come up with a suitable linker-tag which can be attached to thenucleotide and detected by nanopore.

Other examples of suitable tags are:

Fluorescent dyes: Xanthine dyes, Bodipy dyes, Cyanine dyesChemiluminiscent compounds: 1,2-dioxetane compounds (Tropix Inc.,Bedford, Mass.). Amino acids & Peptides: naturally occurring or modifiedaminoacids and polymers thereof. Carbohydrates: glucose, fructose,galactose, mannose, etc. NMPs & NDPs: nucleoside-monophosphates,nucleoside-diphosphates. Aliphatic or aromatic acids, alcohols, thiols,substituted with halogens, cyano, nitro, alkyl, alkenyl, alkynyl, azidoor other such groups.2) Base-Modified Nucleoside-5′-triphosphates

A variety of nucleotide reversible terminators (NRTs) for DNA sequencingby synthesis (SBS) are synthesized wherein a cleavable linker attaches afluorescent dye to the nucleotide base and the 3′-OH of the nucleotideis blocked with a small reversible terminating group [Ju et al. 2006,Guo et al. 2008 & 2010]. Using these NRTs, DNA synthesis is reversiblystopped at each position. After recording the fluorescent signal fromthe incorporated base, the cleavable moieties of the incorporatednucleotides are removed and the cycle is repeated.

The same type of nucleotides can also be used for nanopore DNAsequencing. As shown in FIG. 9(A), a small blocking group at 3′-OH and atag-attached at the base linked through a cleavable linker can besynthesized. After polymerase extension reaction, both the 3′-O-blockinggroup and the tag from the base are cleaved and the released tag can beused to pass through the nanopore and the blockage signal monitored.Four different tags (e.g. different length and molecular weightpoly-ethylenene glycols (PEGs), as shown in FIG. 9(A)) can be used, onefor each of the four bases, thus differentiating the blockage signals.

Alternatively, the 3′-O-blocking group is not used because it has beenshown that a bulky group or nucleotide base can prevent the DNApolymerase from adding more than one nucleotide at a time [Harris et al.2008]. As shown in FIG. 9(B), a bulky dNMP is introduced through acleavable linker. Thus, different dNMPs are introduced through a linkeraccording to the original dNTP. For example, with dTTP nucleotide, adTMP is introduced (for dATP, a dAMP; for dGTP, a dGMP and for dCTP, adCMP is introduced). After polymerase incorporation and cleavage withTCEP, modified dNMPs are generated which are passed through the nanoporechannel and detected by appropriate methods.

3) 2′- or 3′-OH Modified Nucleoside-5′-triphosphates

Synthesis of all four 3′-modified nucleoside-5′-triphosphates can becarried out [Guo et al. 2008, Li et al. 2003, Seo et al. 2004].3′-O-2-nitrobenzyl and 3′-O-azidomethyl attached dNTPs (FIGS. 10A and10B, respectively) are good substrates for DNA polymerases. Afterincorporation by DNA/RNA polymerase in a sequencing reaction, these3′-O-tagged nucleotides terminate the synthesis after single baseextension because of the blocking group at the 3′-OH. Further extensionis possible only after cleavage of the blocking group from the 3′-Oposition. The 3′-O-2-nitrobenzyl group can be efficiently cleaved by UVlight and 2′-O-azidomethyl by treatment with TCEP to generate the freeOH group for further extension. The cleaved product from the reaction(FIG. 10C or 10D) is monitored for electronic blockage by passingthrough the nanopore and recording the signal. Four differentsubstituted nitrobenzyl protected dNTPs and four different azidomethylsubstituted dNTPs, one for each of the four bases of DNA, aresynthesized.

II. DNA-Extension Using Modified Nucleotides 1) Phosphate-TaggedNucleotides

Terminal phosphate-tagged nucleoside polyphosphates described above areused in polymerase reactions to generate extension products. As shown inFIG. 11, after a polymerase reaction, the released by-product of thephosphate-tagged nucleotide, tag-polyphosphate, is obtained and theextended DNA is free of any modifications. The releasedtag-polyphosphate is then used in an engineered nanopore bysingle-channel recording techniques for sequencing analysis. Thereleased tag-polyphosphates can also be treated with alkalinephosphatase to provide free tags which can also be detected. Using fourdifferent tags for the four nucleotides (A, T, G & C) to generate fourdifferent tagged-polyphosphates which differ by mass, charge or bulk,the sequence of the DNA can be determined.

2) Base-Tagged Nucleotides with Cleavable Linkers

Base-tagged nucleotide triphosphates for DNA sequencing by synthesis(SBS) and single molecule sequencing are synthesized [Guo et al. 2008and 2010]. The addition of large bulky groups at the 5-position ofpyrimidines (C & T) and 7-position of 7-deazapurines (G & A) can blockthe addition of more than one nucleotide in a DNA polymerase reaction.Modified nucleotides with a cleavable linker, a bulky group, anddifferent charges attached to the nucleotide base are synthesized. Themodified nucleotides may also have a small blocking group at the 3′-OHof the nucleotides. These modified nucleotides are used in a polymeraseextension reaction. As shown in FIG. 12, after extension with theappropriate nucleotide, the linker and tag from the nucleotide base andfrom sugar 3′-O, if blocked, are cleaved by chemical or photochemicalmeans and the released linker-tag is used in an engineered nanopore bysingle-channel recording techniques for sequencing analysis.

3) 2′- or 3′-Tagged Nucleotides with Cleavable Linkers

A linker and tag can also be attached to the 2′- or 3′-OH ofnucleotides. After a polymerase extension reaction, the linker-tag iscleaved from the extended product by chemical, photochemical orenzymatic reaction to release the free 3′-OH for further extension. Asshown in FIG. 13, the released linker-tag is then used in an engineerednanopore by single-channel recording techniques for sequencing analysis.

III. DNA-Sequencing Study Using Nanopore

Discrimination of different nucleotides in DNA sequencing using nanoporeis evaluated following the strategy shown in FIGS. 11-13. To validate ananopore's ability to distinguish the four different linker-tags in DNA,a series of experiments as shown in FIG. 14 is performed. The DNA/RNApolymerase can be bound to the nanopore and a template to be sequencedis added along with the primer. Either DNA template or primer can alsobe immobilized on top of the nanopore and then subsequently form atemplate-primer complex upon addition of a DNA polymerase. To thistemplate-primer complex, four differently tagged nucleotides are addedtogether or sequentially. After polymerase catalyzed incorporation ofthe correct nucleotide, the added nucleotide releases the tag-attachedpolyphosphate (in case of terminal-phosphate-labeled nucleotides) whichthen pass through the nanopore to generate the electric signal to berecorded and used to identify the added base. Optionally, the releasedtag-polyphosphate can also be treated with alkaline phosphatase toprovide free tag which can also be detected by passing through thenanopore. Each tag generates a different electronic blockade signaturedue to the difference in size, mass or charge. In the case ofbase-modified or 2′/3′-modified nucleotides, after the DNA/RNApolymerase extension, the tag from the extended primer is cleaved bychemical, photochemical or enzymatic means and the electronic signatureof the released tag is monitored. The shape, size, mass, charge or otherproperties of the tag can be adjusted according to the requirements.

As disclosed herein, signals from each of the nucleotides (FIG. 15) andthe transitions between nucleotides of different identities aredistinguished and characterized. The magnitude and duration of theblockade signatures on the event diagram are analyzed and compared withknown diagrams. Thus, with these rational chemical designs andmodifications of the building blocks of DNA, the use of nanopore isoptimized to decipher DNA sequence at single molecule level with singlebase resolution.

To implement this novel strategy for DNA sequencing, an array ofnanopores can be constructed on a planar surface to conduct massiveparallel DNA sequencing as shown in FIG. 18. The array of nanopores canalso be constructed on a silicon chip or other such surfaces. Thenanopore can be constructed from the protein with lipid bilayers orother such layers (α-hemolysin pore, Mycobacterium smegnatis porin A,MspA) [Derrington et al. 2010] or they can be synthetic solid-statenanopores fabricated in silicon nitride, silicon oxide or metal oxides[Storm et. al. 2005; Wanunu et al. 2008] or a hybrid between asolid-state pore and α-hemolysin [Hall et al. 2010].

FIG. 18 shows a schematic of array of nanopores for massive parallel DNAsequencing by synthesis. The nanopores can sense each DNA/RNA polymerasecatalyzed nucleotide addition by-product (Tag-attached to the phosphateor the base and/or 2′, 3′-OH of the sugar moiety) as it passes throughthe nanopore. The electrical properties of different tags willdistinguish the bases based on their blockade property in the nanopores.The array of nanopores shown in FIG. 18 can each read the same sequenceor different sequence. Increasing the number of times each sequence isread will result in better quality of the resulting sequence data.

Example 2

I. Synthesis of PEG-Labeled-Deoxyguanosine-5′-Tetraphosphates(dG4P-PEG):

PEG-labeled-deoxyguanosine-5′-tetraphosphates (dG4P-PEGs) is synthesizedaccording to FIG. 19. First, 2′-deoxyguanosine triphosphate (dGTP)reacts with CDI in DMF to activate the terminal phosphate group which isthen reacted with dibutylammonium phosphate to give the tetraphosphate.The terminal phosphate on this tetraphosphate is further activated withEDAC in 0.1M imidazole buffer followed by reaction with diaminoheptaneto provide an amino attached tetraphosphate which is further reactedwith mPEG-NHS esters to provide the required four PEG-dG4Ps. Afterpolymerase incorporation, the net charge on the released PEG is −3(PEG-NH-triphosphate).

II. Testing of Modified Nucleotides in Single Base Extension Reactions.

The dG4P-PEGs are characterized by MALDI-TOF mass spectroscopy as shownin Table II.

TABLE II MALDI-TOF MS Results for dG4P-PEG Calculated M.W. Measured M.W.dG4P-PEG24 1798 1798 dG4P-PEG37 2371 2374

The dG4P-PEGs are excellent substrates for DNA polymerase in primerextension. The MALDI-TOF mass spectra of the DNA extension products areshown in FIG. 20.

Example 3—Single Molecule Detection by Nanopore of the Pegs Used toLabel the Nucleotides

Poly(ethylene glycol) is a nonelectrolyte polymer that weakly bindscations (e.g., it binds K⁺ ions at K_(d)˜2 M). Thus, the net charge onthe polymer depends on the mobile cation concentration and on thepresence of other moieties that are chemically linked to it. It has beendemonstrated that a single α-hemolysin nanopore can easily distinguishbetween differently-sized PEG polymers at better than monomerresolution, i.e., better than 44 g/mol [Reiner et al. 2010; Robertson etal. 2007]. That level of discrimination is made possible because thepolymer reduces the pore's conductance due to volume exclusion (the poreconductance decreases with increasing polymer size) and by bindingmobile cations that would otherwise flow freely through the pore [Reineret al. 2010]. In addition, the residence time of the polymer in the poreis highly sensitive to the polymer's charge, which for PEG, scales inproportion to the polymer's length. A nanopore should be able todistinguish between differently-sized PEGs that are chemically linked toother moieties. PEGs (PEG 16, 24, 37 and 49) for labeling nucleotidesare tested on nanopore and generate distinct electronic blockadesignatures at the single molecule level as shown in FIG. 21.

To investigate the effect of bulkiness of the variously taggedpolyphosphates on electronic blockade signals generated in the nanopore,various phosphate-linked-nucleotides are synthesized with different sizepolyethylene glycol (PEG) tags attached to the terminal phosphate of thenucleotide. First, as shown in FIG. 4, we synthesize a series ofnucleoside-5′-tri-, tetra-, and penta-phosphates with the terminalphosphate attached via a linker to which different tags, e.g. differentlength and mass PEGs or other molecules to increase the molecular sizeor modify the charge of the released polyphosphate, could be attached.We then test these nucleotides in polymerase reactions coupled withdetection by nanopore to see which tags or bulky groups attached to theterminal phosphate produce more dramatic differences in electronicblockade signals among different bases.

I. Screen and Select 4 PEG Tags with Distinct Nanopore Blockade Signals

Recently, it has been shown that when a polyethylene glycol (PEG)molecule enters a single α-hemolysin pore, it elicits distinctmass-dependent conductance states with characteristic mean residencetimes [Robertson et al. 2007]. FIG. 22A shows that the conductance basedmass spectrum clearly resolves the repeat units of ethylene glycol, andthe residence time increases with the mass of PEG.

I.a Testing PEG for Nanopore Blockade Signatures.

Different length and molecular weight PEGs (commercially available fromQuanta Biodesign Ltd or other suppliers) are selected and the nanoporeblockade signals monitored, as described in Example 2. As shown in FIG.22A PEGs of 28-48 ethylene glycol units are clearly distinguished bynanopore.

Therefore, PEGs with a broad range of ethylene glycol units displayingvery distinct nanopore blockade signals are selected as tags to labelthe nucleotides A, C, G and T. Examples are shown in FIG. 22B. BranchedPEGs as tags are also evaluated as these can be modified with positivecharges in a more straightforward fashion. Structures of some linear andbranched PEGs are shown at the bottom of FIG. 22.

I.b Design and Synthesis of Phosphate-Labeled PEGs Selected in I.a

In nanopore sequencing, the current blockade signals in the nanopore aregenerated by the PEG-phosphates released during the polymerase reaction.Thus, we design and synthesize phosphate-labeled PEGs with positivelycharged linkers, and test these molecules with organic (e.g.,α-hemolysin) and synthetic (solid phase) nanopores to evaluate theircurrent blockade signals. The selected PEGs are converted to theirtriphosphates as shown in FIG. 23. For example, Fmoc-protectedamino-butanol can be converted to the corresponding triphosphate byreacting first with phosphorous oxychloride followed by reaction withtributylammonium pyrophosphate in a one pot reaction. The triphosphateafter purification is activated with DCC/DMF or CDI/DMF to provideactivated triphosphate which reacts with the OH-group of the PEGs togenerate PEG-triphosphates. The same scheme is applicable for bothlinear as well as branched PEGs. These PEG phosphates are tested innanopores to optimize the conditions for generating distinct currentblockade signals.

The polyamino acid (polylysine, polyarginine, interrupted polylysine)linkers are synthesized by standard peptide synthetic strategies; if anester linkage to the polyphosphate chain is built in, it should bepossible to use alkaline phosphatase to cleave it, resulting in morestrongly positive tags for nanopore interrogation. Positive charges mayalso be incorporated into the PEG chains.

I.c Design and Synthesis of a Library of Terminal Phosphate-TaggedNucleoside-5′-triphosphates.

Terminal phosphate tagged nucleoside-5′-tri-, tetra-, andpenta-phosphates are designed and synthesized. These molecules aretested in the polymerase reaction and the optimal ones are selected fornanopore detection. Terminal phosphate-tagged nucleoside-5′-tri-,tetra-, and penta-phosphates with a variety of tags, including small orlarge polylysines, amino acids, a variety of negatively or positivelycharged dyes, such as Energy Transfer dyes, and ethylene glycol units,have been shown to be accepted by DNA polymerases as excellentsubstrates for primer extension [Kumar et al. 2006 and 2008; Sood et al.2005; and Eid et al. 2009].

I.c.1 Design and Synthesis of Terminal Phosphate-TaggedNucleoside-5′-triphosphates.

As shown in FIG. 24, terminal phosphatetagged-nucleoside-5′-triphosphates is synthesized by reacting thecorresponding dNTP with DCC/DMF to yield a cyclic trimetaphosphate whichcan be opened with nucleophiles to generate a tag or linker attachednucleoside-5′-triphosphate. In addition, the linker attached to thephosphate can be reacted with PEG-NHS esters to provide alternatePEG-attached nucleoside-5′-triphosphates. The resulting terminalphosphate-tagged nucleoside-5′-triphosphate is used in thetemplate-primer extension reaction and the released tag-attachedpyrophosphate is detected and differentiated by its specific nanoporecurrent blockade parameters.

I.c.2 Design and Synthesis of Terminal Phosphate-TaggedNucleoside-5′-tetraphosphates.

For synthesis of terminal phosphate taggednucleoside-5′-tetraphosphates, the corresponding triphosphate is firstreacted with CDI in DMF to activate the terminal phosphate group whichis then reacted with phosphoric acid or tag-monophosphate to give thetetraphosphate as shown in FIG. 25. The terminal phosphate on thetetraphosphate can be further activated with EDAC in 0.1M imidazolebuffer followed by reaction with an appropriate nucleophile to provide alinker attached tetraphosphate which can be used to attach tags ofdifferent mass, length or charge, such as m-PEG-NHS esters. In thiscase, four trimethyllysines are used to neutralize the charge of fourphosphates. After polymerase incorporation, the net charge on thereleased PEG is +1 or, if treated with alkaline phosphatase, +4, whichcan be detected by the nanopore.

I.c.3 Design and Synthesis of Terminal Phosphate-TaggedNucleoside-5′-Penta-Phosphates.

Synthesis of terminal phosphate tagged nucleoside-5′-penta-phosphatesfollows the same principle as shown in FIG. 26. They can be preparedeither from activated triphosphates or tetraphosphates by reacting withphosphoric acid, pyrophosphate or tag-attached phosphates.Alternatively, a linker can be attached to the pentaphosphate followedby reaction with activated NHS esters.

The terminal phosphate tagged nucleoside polyphosphates described aboveare used in the polymerase reaction to generate extension products.Following the scheme shown in FIG. 11 the performance of the terminalphosphate tagged nucleoside polyphosphates in polymerase extension areevaluated. We first perform a single base extension reaction andcharacterize the DNA extension product by MALDI-TOF mass spectroscopy toevaluate incorporation efficiency. After establishing optimized reactionconditions, we immobilize the template on magnetic beads and repeat thesingle base extension reaction, after which the releasedpolyphosphate-tags are isolated from the solution for detection using asingle nanopore. This reaction is performed continuously to evaluate all4 nucleotides (A, C, G, and T) and their corresponding released tagsdetected by the nanopore. Continuous polymerase reaction with thepolyphosphate-tag nucleotides and the clear distinction of the releasedpolyphosphate-tag by nanopore establish the feasibility of the approach.

As shown in FIG. 11, after the polymerase reaction, the releasedby-product of the phosphate-tagged nucleotide (tag-polyphosphate) isobtained and the extended DNA strand is free of any modifications. Thisis advantageous because any scars remaining on growing DNA chains canaffect their ability to be recognized by polymerase with increasingnucleotide additions, eventually terminating further DNA synthesis. Thereleased tag-attached polyphosphate are assayed in the nanopore toevaluate sequencing sensitivity and accuracy. In initial experiments wetest the tags for their blockade signals before running SBS reactions.DNA sequence can be determined if different tags for the fournucleotides are used to generate four different tagged-polyphosphateswhich differ by mass, charge or bulk, and yield 4 distinct blockadesignals.

II. Detection of the Released Tagged Phosphates by Protein Nanopores

We use a single α-hemolysin nanopore to detect PEGs that are linked tonucleotides attached via a multi-phosphate linker and the same polymerafter the nucleotide/ribose moiety has been cleaved by the DNApolymerase reaction. Each of the four different DNA bases is linked to aPEG polymer with a unique length. Thus, each base that is removed fromthe PEG by the polymerase is identified. Because the unreactednucleotides cannot be separated from the released tagged polyphosphates,especially in real time situations, we take advantage of the method'sextreme sensitivity to molecular charge to discriminate between thereleased reaction product and the starting material. We measure singleα-hemolysin conductance using conical glass supports [White et al., 2006and 2007] which allow data collection at 100 kHZ and ˜4 pA RMS noise. Wemeasure the blockade depth and residence time distributions of both thetagged nucleotides and tagged products over a wide range oftransmembrane potentials to determine optimum conditions for nucleotidediscrimination and to extend our current theoretical understanding ofPEG-nanopore interactions [Robertson et al. 2007] to molecules withfixed charges. Characterization and theoretical understanding permit theunambiguous identification of the nucleotides incorporated intopolynucleotides by polymerase. Thus, with these rational chemicaldesigns and modifications of the building blocks of DNA, we optimize theuse of nanopores to decipher DNA at the single molecule level withsingle base resolution in protein or synthetic nanopores.

Example 4—Fabrication of a Single Solid-State Nanopore for SingleMolecule Sequencing

The transition from a protein nanopore to a solid state nanopore makesthe fabrication of high-density nanopore arrays possible, a key step foryielding a high-throughput single molecule electronic DNA sequencer.Here, an integrated single solid state nanopore platform is developed tocharacterize the tagged nucleotides in the polymerase reaction based onthe knowledge gained from the protein nanopore.

Integrated Nanopore Platform.

We developed specialized integrated low-noise CMOS electronics, whichwhen integrated with solid-state nanopores, deliver significantperformance advantages over “standard” measurement techniques whichemploy external electrophysiological amplifiers, such as the Axopatch200B. These advantages come from exploiting capacitive (rather thanresistive) feedback in a custom integrating amplifier design. DCcurrent, which is characteristic of this and other bioelectronicinterfaces, is removed with a low-noise current source operating in a DCservo loop. Reduced amplifier input capacitances and reduced parasiticcapacitances associated with co-integration improve noise performance athigh frequencies, enabling bandwidths approaching 1 MHz for solid-statepores. Such high temporal resolution, when combined with the tagsdeveloped, will provide high flexibility for tuning this platform forhigh sensitivity and real-time performance.

Use of this CMOS-integrated nanopore (CNP) integrated circuit in eithera two-chip or one-chip configuration as shown in FIG. 27. In the formercase, the pore is packaged together with the CNP as shown in FIG. 27B.In the latter, the pore is fabricated directly into the CNP as shown inFIG. 27C with fluidics on either side of the chip. In both cases, thecis electrode, which connects to the input of the amplifier, isintegrated directly on the surface of the CNP. The one-chipconfiguration has the advantage of being easily scalable to amultiplexed platform at the cost of additional fabrication complexity.The ability to post-process fabricated CMOS dice (which are no more than5 mm on a side) is a unique capability established over the last fiveyears [Huang et al. 2011, Lei et al. 2008, and Levine et al. 2009]. Thisapproach completely leverages existing foundry process flows rather thanrequiring new process development.

The one-chip fabrication approach proceeds by adapting standardsolid-state nanopore fabrication techniques [Rosenstein et al. 2011]. Inareas of the die reserved for the sensors, all metals have been blocked,leaving a thick stack of alternating glass fill and silicon nitridecapping layers. The majority of the dielectric stack is etched using aninductively-coupled CHF₃ plasma. After depositing and patterning a PECVDSi₃N₄ etch mask on the back of the die, localized openings in thesilicon substrate are made using an anisotropic potassium hydroxideetch. A short dip in buffered hydrofluoric acid is then used to isolatea single 50 nm layer of silicon nitride from the original dielectricstack as a suspended membrane. Finally, nanopores are drilled throughthese nitride membranes with a high resolution transmission electronmicroscope.

The measured noise of this system is shown in FIG. 28A, alongside ameasurement of the baseline noise for a comparable configuration of theAxopatch 200B. For the highest bandwidth supported by the Axopatch(B=100 kHz), the integrated amplifier has a noise floor of 3.2 pA_(RMS),compared to 9 pA_(RMS) for the Axopatch. At the highest bandwidthcharacterized for the integrated amplifier (B=1 MHz), the noise level is27 pA_(RMS), in contrast with 247 pA_(RMS) modeled by extrapolating theAxopatch response beyond its supported range (approximately afactor-of-ten lower noise). As a point of comparison, for a 1 nA signal,only about 6250 ions are transported through the pore in 1 us. Aninput-referred noise level of 27pAV_(RMS) for integrated amplifierallows resolution of as few as 150 ions in this interval.

It is also important to note this superior electrical performance isobtained with an integrated amplifier that consumes an area of only 0.2mm² on a CMOS chip compared with a rack-mounted Axopatch amplifier,demonstrating the significance of the innovative electronics. When ananopore is connected to the amplifier input, the introduction of 1/fnoise and membrane capacitance raises the noise spectrum above theopen-headstage baseline. FIG. 28b shows a typical noise spectrum in thiscase, demonstrating noise floors of only 10 pA_(RMS) and 163 pA_(RMS)for bandwidths of 100 kHz and 1 MHz, respectively. Measured comparisonsare shown with the Axopatch up to 100 kHz for the same nanopore. At 100kHz, there is more than a factor-of-two reduction in input-referrednoise power for the CNP. If the Axopatch could be measured at higherbandwidths, there would be a factor-of-six noise power difference at 1MHz.

This platform also allows the integration of biological nanopores,providing even more flexibility. Biological nanopores are created inlipid membranes (typically 1, 2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)) formed over a hole in a teflon membrane between two fluid cells.The surface must be sufficiently hydrophilic for the membrane to formfrom unilamellar vesicles. The conductance between the two chambers ofthe cell is monitored while the membrane protein is added to one of thecells, which is immediately flushed once incorporation is detected. Themembranes used to fabricate the nanopores can also be used as solidsupports for lipid bilayers with the drilling of larger holes into themembranes, over which the lipid bilayer is formed [Clarke et al. 2009;Benner et al., 2007; Hou et al., 2009; and Wang et al. 2011]. Planarbilayer lipid membranes (BLMs) have been engineered with differentprotein channels on patterned solid supports with nanopatterned holes(˜100 nm in diameter), as well as tethering them directly on goldthrough a self-assembled monolayer assembly [Axelrod et al., 1976,Bultmann et al. 1991, Dutta et al. 2010, Jenkins et al. 2001, Nam et al.2006, Palegrosdemange et al. 1991, Shen et al. 2009, Srinivasan et al.2001, Yang et al. 2003, Yin et al. 2005]. Moreover, it has been shownthat formation of contiguous BLMs with a diffusion coefficient of 4μm²/s on nanopatterned substrates; BLMs formed on SAM-gold assembliesyielded a coefficient of 0.8 μm²/s. Both fall within the ideal diffusionrange of 0.1-10 μm²/s representative of well-formed BLMs [Axelrod et al.1976, Bultmann et al. 1991]. Electrical characterizations of these BLMsindicate a high impedance membrane with a 1.4GW-mm² resistance, makingit amenable for further electrical analysis of biological nanoporesformed in the membrane [Oliver et al. 1994, Shi et al. 2000, Wiehelman1988].

Immobilization of Polymerase to Nanopore-Bearing Surfaces

The size of the polymerase is about 5 nm×5 nm. One polymerase ispositioned near the entrance to each nanopore. To accomplish this forthe solid-state nanopores, it is necessary that (1) a unique position onthe surface be modified with functional groups during CMOS fabricationto bind the polymerase; (2) that the sites be small enough that only onepolymerase molecule can bind; (3) that they be far enough apart thatthere is little possibility of diffusion of the released taggedpolyphosphates to a nearby channel; and (4) that the cross-linking agentbe sufficiently flexible that the enzyme is functionally intact.Polymerase tethering is accomplished by combining a patterned attachmentpoint with the use of an appropriate concentration of polymerasesolution during incubation such that at most one enzyme molecule isattached.

Establishment of the appropriate tether point for the polymerase isaccomplished by exploiting existing fabrication approaches forsolid-state nanopores. Typically, to maximize the transduction signals,these pores are created by thinning a supported Si₃N₄ membrane usinge-beam lithography to define a window which is subsequently thinned witha plasma etch (e. g. SF₆). The nanopore is then drilled in the thinnedregion using e-beam ablation. The well created by this window (FIG. 29)creates a natural place to tether the polymerase, guaranteeing closeproximity to the nanopore entrance. Prior to etching the thinned window,the original membrane can be augmented with a buried epitaxial layer ofattachment material. Once the window is etched, this can become aselective sidewall region for polymerase attachment. Attachmentmaterials include silicon dioxide or gold. There may be limitedselectivity with silicon dioxide, however, because an oxide can alsoform on the silicon nitride surface under appropriate conditions.

In principle, with silicon dioxide surfaces, biotin-streptavidinlinkages can be used [Korlach et al. 2008 and 2010], utilizingbiotinylated PEG molecules on the silica patches and incubate biotin-endlabeled polymerase in the presence of streptavidin. The remainder of thesurface is passivated with polyvinylphosphonic acid. Due to the concernsraised above, it is preferable instead to modify the gold surface withan alkanethiol self-assembled monolayer (SAM) functionalized with aminogroups [Love et al. 2005]. These can be easily modified to NHS estersfor attachment to amino groups on the polymerase. The thickness andhomogeneity of the layer is determined by ellipsometry or atomic forcemicroscopy.

Development of 5′-Modified Nucleotides with Positively Charged Linkers

A system for rapid diffusion of the released tags toward the pores whilethe precursor nucleotides and DNA are repelled by the pores isgenerated. The tagged nucleotides are engineered so that afterincorporation into the DNA, the tag released from the nucleoside has acumulative positive charge while the intact tag-nucleotides remainneutral. This allows actively gating the released tag specificallythrough the detection channel, if the channel is negatively chargedaccording to known methods [Wanunu et al. 2007]. As all other freemolecules present in the reaction mix (primers, unreacted nucleotides,template), other than the tag, are negatively charged, only the releasedtag carrying positive charge is attracted into the channel, increasingthe specificity of detection and reducing noise. A different number ofcharged groups can be used on different tags, depending on the specificnucleotide base. Thus the cumulative charge of the tag along with itssize can be used for base discrimination. After incorporation andrelease of the tag, if the polyphosphate is deemed to mask the positivecharge, it can be removed using secondary reactions (for example,alkaline phosphatase immobilized at a second downstream site in thepore). The positively charged tag can be gated into the negativelycharged channel for detection and recognition.

Diffusion and Drift

A critical aspect of this sequencing system is the reliable and timelycapture of each nucleotide's released tag by the adjacent nanopore.Conditions must be engineered such that tags are captured quickly and inthe correct order. Additionally, the capture rate of unincorporated tagsshould be minimized, and interference from adjacent channels should benegligible. Creating the well at the entrance of the pore (as shown inFIG. 29) assists this process, which also depends on close proximity ofthe polymerase to the nanopore opening. Analysis of nanopore captureprocesses generally considers a radially symmetric process surroundingthe pore. Geometry dictates that in the absence of an electric field, amolecule tends to diffuse farther from a pore, opposing theelectrostatic attraction. With a voltage gradient, there exists acritical distance L at which molecular motion due to diffusion andelectrophoresis are equal [Gershow et al. 2007]. This critical distanceis a function of the ionic current (I) and electrolyte conductivity (σ),as well as the diffusion constant (D) and mobility (φ of the analytemolecule,

$L = {\frac{l}{2{\pi\sigma}}{\frac{\mu}{D}.}}$

Capture is a statistical process, but approximately 50% of molecules ata distance L is captured. This likelihood increases for shorterdistances, and exceeds 90% for d<L/3. During this process, moleculestypically are captured in a timescale on the order of

$t_{capture} = {{\frac{L^{3}}{s\text{?}}.\text{?}}\text{indicates text missing or illegible when filed}}$

By placing the polymerase within L/3 of the nanopore, nearly allmolecules are captured. It also ensures that t_(capture) issignificantly faster than the polymerase incorporation rate, to capturebases in the correct order.

An approximate value for the diffusion coefficient of 25-unit PEGmolecules in water is D=3e-10 m²/s [Shimada et al. 2005], which is onthe same order of magnitude as a similar-length ssDNA fragment [Nkodo etal. 2001]. Assuming validity of the Nernst-Einstein relation (althoughthis does not always hold true for polymers), the mobility can beestimated as a function of the diffusion constant and net charge (Q),

$\mu \approx {\frac{QD}{k_{B}T}.}$

For these estimates, then, with I=5 nA in 1M KCl—see the followingTable.

+1e +4e 50% capture 2.1 nm  5.8 nm 90% capture 0.7 nm  1.9 nmt_(capture) 7.1 ns 114 ns

Example 5—Fabricate an Array of Solid-State Nanopores

In addition to improved performance, only with the integratedelectronics is it possible to produce massively parallel nanoporearrays. This involves the one-chip topology shown in FIG. 27C in whichnanopores are integrated directly into the CMOS die with fluidics oneither side of the chip. The approach for integrating multiple pores isalso shown in FIG. 27C. In this case, wells of SU-8 photoresist are usedto isolate individual nanopores from each other. This is an approachsimilar to that of Rothberg et al. 2011. In Rothberg et al., however,the wells can still remain “connected” by the solution reservoir abovethe chip. In present case, since electrical isolation is necessarybetween the cis reservoirs, a PDMS cap is used to seal the wells formeasurement after the introduction of reagents as shown in FIG. 27C. 64solid-state nanopores are integrated onto the same 5-mm-by-5-mm die. Thecurrent integrating amplifier design, which would have to be duplicatedat each pore site, is only 250 um by 150 um, but additional space has tobe left for the fabrication of the pore itself. As fabricationtechniques are further developed to reduce the chip area, this can beeasily scaled to an array of 16-by-16 electrodes.

Example 6—Pyrosequencing Using Phosphate-Tagged Nucleotide and NanoporeDetection

Pyrosequencing is sequencing by synthesis (SBS) method which relies onthe detection of pyrophosphate that is released when a nucleotide isincorporated into the growing DNA strand in the polymerase reaction[Ronaghi et al. 1998]. In this approach, each of the four dNTPs is addedsequentially with a cocktail of enzymes, substrates, and the usualpolymerase reaction components. If the added nucleotide is complementaryto the first available base on the template, the nucleotide will beincorporated and a pyrophosphate will be released. Through an enzymecascade, the released pyrophosphate is converted to ATP, and then turnedinto a visible light signal by firefly luciferase. On the other hand, ifthe added nucleotide is not incorporated, no light will be produced andthe nucleotide will simply be degraded by the enzyme apyrase.Pyrosequencing has been applied successfully to single nucleotidepolymorphism (SNP) detection and DNA sequencing. A commercial sequencingplatform was developed combining pyrosequencing and DNA templateamplification on individual microbeads for high-throughput DNAsequencing [Margulies et al. 2005]. However, there are inherentdifficulties in pyrosequencing for determining the number ofincorporated nucleotides in homopolymeric regions (e.g. a string ofseveral T's in a row) of the template. Beside this, there are otheraspects of pyrosequencing that still need improvement. For example, eachof the four nucleotides has to be added and detected separately. Theaccumulation of undegraded nucleotides and other components could alsolower the accuracy of the method when sequencing a long DNA template.

This is a modified pyrosequencing approach which relies on the detectionof released tag- or tag-phosphates during polymerase reaction. In thisapproach, phosphate-tagged nucleotides are used in polymerase catalyzedreaction on a template-primer complex. Upon incorporation of thetagged-nucleotides, the phosphate-tag moiety is released, which can bedetected by passing through a nanopore. The same tag can be used on eachnucleotide or a different molecular weight and length tag (such as PEGs)can be used. It has been shown that polyethylene glycols (PEGs) ofdifferent length and mass can be resolved at single-molecule sensitivitywhen passed through hemolysin nanopore [Robertson et al. 2009].

An α-hemolysin channel could be used to detect nucleic acids at thesingle molecule level [Kasianowicz et al. 1996]. The monomericpolypeptide self-assembles in a lipid bilayer to form a heptameric pore,with a 1.5 nm-diameter limiting aperture. In an aqueous ionic saltsolution, the pore formed by the α-hemolysin channel conducts a strongand steady ionic current when an appropriate voltage is applied acrossthe membrane. The limiting aperture of the nanopore allows linearsingle-stranded but not double-stranded nucleic acid molecules (diameter˜2.0 nm) to pass through. The polyanionic nucleic acids are driventhrough the pore by the applied electric field, which blocks or reducesthe ionic current. This passage generates a unique electronic signature.Thus a specific event diagram, which is the plot of translocation timeversus blockade current, will be obtained and used to distinguish thelength and the composition of polynucleotides by single-channelrecording techniques based on characteristic parameters such astranslocation current, translocation duration, and their correspondingdispersion in the diagram. Four PEG tags, which have been shown to yielddistinct current blockade signals in nanopores, are selected to couplewith four nucleotides (A, C, G, T) at the terminal phosphate. Thesenovel nucleotide analogs are used in a polymerase reaction and usenanopores to detect the released tags for decoding the incorporatedbases as shown in FIG. 14.

There are several advantages to this approach:

-   -   1) Avoid the use of many different enzymes (saves cost and        complexity).    -   2) Addition of single tag-attached nucleoside polyphosphate        sequentially or all four nucleotides with different tags        attached to each nucleotide.    -   3) Use of PEGs as tags which can be detected by nanopore at a        single unit resolution.    -   4) Real time Single molecule detection sequencing as the tag        passes through the nanopore.    -   5) Massively parallel sequencing, low cost and high throughput.

As shown in FIG. 14, DNA polymerase is immobilized to the nanopore andthe template-primer along with the PEG-tagged nucleotides is added. Onincorporation of the correct PEG-tagged nucleotide, the releasedPEG-phosphates pass through the nanopore and the electronic blockadesignal is measured. Different length PEGs have different blockadesignals, thus, 4 different PEGs can be used for 4 different nucleotides.

The nucleotides can be added one at a time, if the correct nucleotide isadded it gives a distinct blockade signal. However, if the nucleotide isnot complementary to the template nucleic acid base, it will not beincorporated and thus no signal detected. In a massive parallel way highdensity array of micro/nano wells to perform the biochemical process canbe constructed. Each micro/nano-well holds a different DNA template andnanopore device. The released PEGs are detected at single-moleculesensitivity.

General methods for synthesis of TAG-labeled-nucleoside-5′-polyphosphateis shown in FIG. 30. Terminal-phosphate-labeled-nucleoside-5′-tri,tetra-, penta-, or hexa-phosphates can be synthesized starting from thecorresponding nucleoside-5′-triphosphates (NTP). Thus, triphosphate isfirst activated with DCC/DMF which can be directly reacted with theTAG-nucleophile to give TAG-attached-NTP or it can be reacted with alinker nucleophile to which a TAG-NHS or appropriately activated TAG canbe reacted to provide TAG-linker-attached NTP. For the synthesis ofTAG-attached nucleoside tetraphosphates (N4P) or pentaphosphates (N5P),the activated triphosphate is first reacted with phosphoric acid orpyrophosphate to give tetra- and penta-phosphate, respectively, whichcan be reacted with linker nucleophile followed by the reaction withappropriate activated TAGs.

Synthesis of PEG-labeled nucleotides are discussed above in Examples 2and 3. The PEG-labeled nucleotides have −3, −4, −5, or −6 charges basedon the use of tri, tetra-, penta-, or hexa-phosphates. After polymerasecatalyzed primer-extension reaction, the net charge on the releasedPEG-tags will be one less (−1) than the starting PEG-nucleotide which isenough to distinguish by the nanopore ionic blockade signal (unreactedPEG-nucleotide is also bulkier than the released PEG-phosphates, thusdifferent ionic blockade signal). Alternatively, if alkaline phosphataseis present in the reaction mixture, the released PEG will be neutral(the free phosphate groups are hydrolyzed by alkaline phosphatase). Thereleased PEG-tags can also be made positively charged as shown below sothat they can be easily detected by nanopores. Similarly, they can alsobe made highly negatively charge.

Synthesis of Positively Charged TAG-Attached-Nucleoside-Polyphosphates:

The positively charged TAG-attached nucleoside-polyphosphates aresynthesized as shown in FIG. 25. First, a positively chargedtrimethyl-(lysine)_(n)-glycine amino acid (K[(Me)₃]_(n)-Gly) is reactedwith the PEG-NHS ester and then activated to form thePEG-K[(Me)₃]_(n)-Gly-NHS ester. This activated ester is reacted with theamino-terminated nucleoside-polyphosphate as shown in FIGS. 19 and 25.The net charge on the nucleoside-tetraphosphate is neutral but afterpolymerase incorporation, the released PEG has a +1 positive charge andif alkaline phosphatase is added to the reaction cocktail, the netcharge on the released PEG is +4. Thus the released TAG can be easilyseparated and identified by passing through the nanopore.

Synthesis of 3′-Blocked-PEG-Attached-Nucleoside-Polyphosphates forSequencing by Synthesis with Nanopore Detection.

The synthesis of 3′-blocked-nucleoside-polyphosphates essentiallyfollows the same route as shown for TAG-attachednucleoside-polyphosphates, except that the startingnucleoside-5′-triphosphate is 3′-O-blocked-dNTP. As shown in FIG. 31,3′-O-azidomethyl-dNTP (6) is first reacted with CDI or DCC/DMF followedby reaction with phosphoric acid (tetraphosphate) or pyrophosphate(pentaphosphates). This is reacted after purification with theappropriate nucleophile to provide amino-terminated phosphate which isthen reacted with the appropriate PEG-NHS ester (neutral, positivelycharged or negatively charged) to provide required3′-O-blockade-PEG-attached-nucleoside-polyphosphate.

Sequencing Scheme with PEG-Nucleotides and Nanopore Detection(many copies of a DNA molecule are immobilized on a bead and sequentialaddition of one PEG-nucleotide at a time).

As shown in FIG. 32, the DNA molecules are immobilized on a bead. Thuseach bead has many copies of the same DNA molecule. The bead is added toa micro/nano-well which is attached to a nanopore. The DNA forms thecomplex with the DNA polymerase which is either attached to the nanoporeor added to the micro/nano well along with the PEG-attached nucleotide.The nucleotides can be added one at a time, if the correct nucleotide isadded it is incorporated and release a PEG-Tag which gives a distinctblockade signal when passed through a nanopore. However, if thenucleotide is not complementary to the template nucleic acid base, itwill not be incorporated and thus no signal detected. In this case, thesame length and molecular weight PEG can be used on all fournucleotides, or, if desired, four different PEGs can also be used. Thus,addition of nucleic acid base can be easily detected by the nanoporeblockade signal at single-molecule sensitivity.

Sequencing by Synthesis with 3′-O-Blocked-PEG-Nucleotides and NanoporeDetection(many copies of a DNA molecule are immobilized on a bead andsimultaneous addition of all four 3′-O-blocked-PEG-nucleotides).

The homopolymeric regions of the DNA can be corrected sequenced usingthis approach. Thus, if the 3′-OH group of the nucleotide is blocked bya reversible moiety, the DNA synthesis will stop after addition of onlyone nucleotide. The synthesis can be continued after the removal of theblocking group to generate a free 3′-OH group. As shown in FIG. 33 allfour different size PEG-attached-3′-O-azidomethyl-nucleotides can beadded to the reaction micro/nano-well and whenever a correct nucleotideis incorporated, the released PEG-tag is read by passing through thenanopore and ionic signal detected. Because 3′-OH group is blocked onlyone nucleotide is added at one time. This 3′-O-blocked group can becleaved by TECP treatment and thus free OH group is ready for furthernucleotide incorporation. By repeated nucleotide addition and cleavage,homopolymeric region can be correctly and easily sequenced.

Massively Parallel Pyrosequencing Using Nanopores:

As shown in FIG. 34, in a massive parallel way high density array ofmicro wells to perform the biochemical process can be constructed. Eachmicro/nano-well holds a different DNA template and nanopore device. Thereleased PEGs are detected at single-molecule sensitivity.

Summary of Experiment

-   -   1) Any TAG of different size, length, molecular weight, charge        attached to the terminal phosphate of the nucleotide which can        be detected by nanopore after polymerase incorporation.    -   2) TAG attached to the tri-, tetra-, penta-, hexa-phosphates.    -   3) Electronic Detection    -   4) Group of DNA molecules attached to the bead or solid surface        and single-molecule detection sensitivity (High density and high        sensitivity).    -   5) Easily sequenced homopolymeric region by using        TAG-attached-3′-O-blocked nucleotides.    -   6) Add one TAG-nucleotide per cycle.    -   7) Add all four reversibly tagged-nucleotides together for        sequencing homopolymeric regions.    -   8) High sensitivity, accuracy and speed.    -   9) Massive parallel sequencing.

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1.-86. (canceled)
 87. A method for determining sequence informationabout a template nucleic acid molecule, comprising: (a) providing asubstrate having at least one nanopore, the at least one nanopore havinga top opening and a bottom opening, and having a single polymeraseenzyme attached proximal to an opening, the polymerase enzyme complexedwith a primed template nucleic acid; (b) contacting the substrate with asequencing reaction mixture comprising reagents required for polymerasemediated nucleic acid synthesis including two or more different types ofnucleotide analogs, each comprising a current blockade label attached tothe phosphate portion of the nucleotide analogs such that the currentblockade label is cleaved upon incorporation of the nucleotide into agrowing strand; (c) providing a voltage drop across the nanopore suchthat the current blockage label produces a change in current through thenanopore; (d) measuring the current through the nanopore over time todetect the incorporation of nucleotides into the growing strand; and (e)identifying the type of nucleotide incorporated into the growing strandusing current blockage characteristics, thus determining sequenceinformation about the template nucleic acid molecule.
 88. The method ofclaim 87, wherein the sequencing reaction mixture comprises fourdifferent types of nucleotide analogs, each corresponding to the basesA, G, C, and T, or A, C, G, and U.
 89. The method of claim 88, whereineach of the types of nucleotide analogs has a different blockade label.90. The method of claim 87, wherein the current blockage characteristicscomprise the magnitude of the current through the nanopore.
 91. Themethod of claim 87, wherein the current blockage characteristicscomprise the shape of the measured current through the nanopore overtime.
 92. The method of claim 87, wherein the nucleotide analogs havethe structure NS-PP-L-B wherein NS comprise a nucleoside moiety, PPcomprises a polyphosphate chain with at least two phosphates, Lcomprises a linker, and B comprises a charge blockade label.
 93. Themethod of claim 87, wherein the different types of nucleotide analogshave different current blockade labels, each having a different level ofnet charge.
 94. The method of claim 87, wherein the different types ofnucleotide analogs comprise linkers having different lengths.
 95. Themethod of claim 87, wherein a blockade label comprises a protein. 96.The method of claim 87, wherein a blockade label comprises lysine orarginine.
 97. The method of claim 92, wherein the linker comprisespolyethylene glycol or a branched or linear alkane.
 98. The method ofclaim 87, wherein the phosphate portion of a nucleotide analog comprises3, 4, 5, 6, or 7 phosphates.
 99. The method of claim 87, wherein thenanopore comprises a solid state nanopore.
 100. The method of claim 87,wherein the nanopore comprises a nanopore protein.
 101. The method ofclaim 87, wherein the nanopore comprises a hybrid nanopore comprising asolid-state pore dimensioned and treated so as to accept a singlenanopore protein.
 102. The method of claim 87, wherein the polymeraseenzyme is attached directly to the substrate.
 103. The method of claim87, wherein the voltage drop across the nanopore is positive on the topside of the nanopore relative to the bottom side of the nanopore, suchthat positively charged molecules tend to be transported through thenanopore.
 104. The method of claim 103, wherein the cleaved portions ofthe nucleotide analogs have a net negative charge.
 105. The method ofclaim 87, wherein the current is measured using measurements ofresistance, or impedance.
 106. The method of claim 87, wherein each ofthe nucleotide analogs comprise a linker between the phosphate of thenucleotide analog and blockade label, whereby when the base portion ofthe nucleotide analog is associated with the polymerase enzyme while itis incorporated into a growing strand during nucleic acid synthesis, thecurrent blockage label at least partially enters the nanopore, resultingin a measurable change in current through the nanopore while thenucleotide analog is associated with the polymerase, and using thechange in current while the nucleotide analog is associated with thepolymerase to detect and identify the incorporated nucleotide.